Method of inducing immune tolerance

ABSTRACT

Methods for inducing tolerance to a transplant in a subject are disclosed. The methods comprise administering multiple doses of a therapeutically effective amount of a CD40 antagonist alone or in combination with a CD86 antagonist, wherein the first dose of the antagonist is given before or at the time of transplantation; and administering multiple doses of a therapeutically effective amount of an immunosuppressive drug, wherein the first dose of the immunosuppressive drug is given at least several days after transplantation.

BACKGROUND OF THE INVENTION

The first step leading to the initiation of an immune response is therecognition of antigen fragments presented in association with majorhistocompatibility complex (MHC) molecules. Recognition of antigens canoccur directly when the antigens are associated with the MHC on thesurface of foreign cells or tissues, or indirectly when the antigens areprocessed and then associated with the MHC on the surface ofprofessional antigen presenting cells (APC). Resting T lymphocytes thatrecognize such antigen-MHC complexes become activated via association ofthese complexes with the T cell receptor (Jenkins et al., J. Exp. Med.165, 302-319, 1987; Mueller et al., J. Immunol. 144, 3701-3709, 1990).If T cells are only stimulated through the T cell receptor, withoutreceiving an additional costimulatory signal, they become nonresponsive,anergic, or die, resulting in downmodulation of the immune response, andtolerance to the antigen. (Van Gool et al., Eur. J. Immunol.29(8):2367-75, 1999; Koenen et al., Blood 95(10):3153-61, 2000).However, if the T cells receive a second signal, termed costimulation, Tcells are induced to proliferate and become functional (Lenschow et al.,Annu. Rev. Immunol. 14:233, 1996).

Activated T cells express high levels of CD154 (CD40L). The cell surfaceexpression of CD154 is tightly regulated and its biological activity ismediated by binding of the extracellular region of CD154 with CD40 onAPC. In normal allogeneic recognition, CD154/CD40 interaction leads toupregulation of the B7 molecules, CD80 and CD86, Class I and Class IIMHC, as well as various cytokines (Caux et al., J. Exp. Med. 180:1263,1994) resulting in additional T cell activation, B cell proliferationand induction of antibody secretion. Therefore, the CD154/CD40interaction can be considered as a major costimulatory signal for theactivation of immune responses.

Members of the B7 family of proteins, B7-1 (CD80) and B7-2 (CD86),expressed on APCs are also critical costimulatory molecules (Freeman etal., J. Exp. Med. 174:625, 1991; Freeman et al., J. Immunol. 143:2714,1989; Azuma et al., Nature 366:76, 1993; Freeman et al. Science 262:909,1993). CD86 appears to play a predominant role during primary immuneresponses, while CD80, which is upregulated later in the course of animmune response, may be important in prolonging primary T cell responsesor costimulating secondary T cell responses (Bluestone, Immunity 2:555,1995). Moreover, the receptor to which a B7 molecule binds, such as CD28or an inhibitory receptor such as CTLA-4, dictates whether the resultingsignal to the immune cell is costimulation or inhibition. Both CD80 andCD86 exhibit binding affinity for both the costimulatory receptor CD28and the inhibitory receptor CTLA4 (CD152). CD28 is constitutivelyexpressed on the majority of T cells, and binding of CD86 and/or CD80 tothis receptor induces the expression of anti-apoptotic proteins,stimulates growth factor and cytokine production and promotes T cellproliferation and differentiation. In contrast, CD152 is only expressedfollowing T cell activation (Brunet, J. F., et al., 1987 Nature 328,267-270), and the interaction of CD86 and CD80 with CD152 appears to becritical for the down-regulation of T cell responses (Waterhouse et al.,Science 270:985, 1995; Allison and Krummel, Science 270:932, 1995).Further, the different expression patterns of the two receptors throughthe course of T cell activation is thought important for appropriateregulation of the T cell response, since the B7 molecules have a higheraffinity for CD152 than for CD28 (Linsley, P. S., et al., 1991 J. Exp.Med. 174, 561-569). Thus, low CD80/CD86 expression levels results inCD152 ligation and dampening of T-cell responses, while high expressionlevels of CD80/CD86 results in ligation to both CD152 and CD28 resultingin T cell activation and costimulation.

Current clinical strategies for general long-term immunosuppression indisorders associated with an undesired immune response (e.g., graftrejection) are based on the long-term administration of broad actingimmunosuppressive drugs, for example, signal 1 blockers such as forexample cyclosporin A (CsA), FK506 (tacrolimus) and corticosteroids.However, while these immunosuppressive regimens have led to a dramaticreduction of the incidence of acute rejection episodes, they have yet toachieve a similar effect for chronic rejection or chronic/sclerosingallograft nephropathy (CAN), which is still the leading cause of graftloss during long-term follow-up. In addition, the high doses of thesedrugs required immediately after transplantation can be toxic to manypatients leading to damage of the transplanted tissue or organ. Inaddition, long-term use of high doses of these drugs can also have toxicside-effects. Moreover, even in those patients that are able to toleratethese drugs, the requirement for life-long immunosuppressive drugtherapy carries a significant risk of severe side effects, includingtumors, serious infections, nephrotoxicity and metabolic disorders (Penn2000; Fishman et al. 1998).

A number of recent studies have explored the effects of antibodies andfusion proteins that bind to various members of the B7 family and/ortheir ligand molecules on the induction of tolerance in allograftrecipients. For example, it was recently demonstrated in non-humanprimates that a combination of anti-CD80/CD86 treatment in renalallograft recipients does not lead to the induction of tolerance. Whiletreatment with the murine CD80/CD86 antibodies prolonged graft survival,even humanized monoclonal antibodies were not able to induce stabletolerance in all recipients (Ossevoort et al., 1999; Kirk et al. 2001;Hausen et al. 2001). Treatment with CTLA4-Ig also blocked CD80 and CD86,but was not very effective in prolonging graft survival (Kirk et al.1997).

A number of other studies have examined the effects of antibodies toCD40 and/or CD154 on activation of the immune system. For example, theuse of a humanized antagonistic anti-CD154 mAb (hu5c8) alone, incombination with CTLA4-Ig, or in combination with anti-CD80 and CD86antibodies in rhesus kidney (Kirk et al 1997, Kirk et al. 1999), rhesusheart (Pierson et al. 1999), rhesus pancreatic islet transplantation(Kenyon et al 1999-a) or pancreatic islet transplantation in baboons(Kenyon et al 1999-b). These studies led to long survival times in mostmonkeys, in many cases long after cessation of treatment. Long-termkidney allograft recipients treated with hu5c8 alone lost theirdonor-specific MLR reactivity, but remained capable of formingdonor-specific antibodies and graft infiltrating lymphocytes (Kirk et al1999). However, trials with hu5c8 in human renal allograft recipientswere aborted after reports of thromboembolic events in autoimmunestudies conducted simultaneously (Knechtle et al. 2001), as activatedplatelets also express CD154. In addition, the hu5c8 seemed lesseffective in human kidney recipients than in non-human primates.

Recently, it was observed that treatment of rhesus monkey kidneyallograft recipients with a combination of anti-CD80, anti-CD86 andanti-CD154 delayed the development of anti-donor antibodies, althoughsurvival times were not significantly prolonged over anti-CD154treatment alone (Montgomery et al. 2001). Another recent study reportedthat blocking costimulation by anti-CD40 or anti-CD40 plus anti-CD86prevented graft rejection in rhesus monkey allograft recipients for theduration of the treatment, but was unable to sustain graft acceptanceonce treatment was terminated (Haanstra et al. 2003).

Accordingly, there is a need for improved therapeutic approaches thatare capable of efficiently inducing long-term immune tolerance to graftswithout the need for administration of high initial doses ofimmunosuppressive drugs, such as signal 1 blockers, that are toxic tomany patients. The successful induction of immune tolerance wouldfurther obviate the need for long-term administration immunosuppressivedrugs, thereby reducing not only the costs of such treatments, but alsothe risk of cancer and infection in graft recipients subjected tolong-term immunosuppressive therapies.

SUMMARY OF THE INVENTION

The present invention provides improved therapies for inducing toleranceto a transplant in a subject, without the need for initialadministration of toxic immuno-suppressive drugs. Immune tolerance isinduced by administering a CD40 antagonist, alone or in combination withan antagonist to another costimulatory molecule (e.g., CD86), followedby administration of immunosuppressive drugs to inhibit T cellcostimulation and thereby induce T cell tolerance. Using this treatmentregimen, long-term tolerance beyond that previously achieved, andpreferably in the absence of continued immunosuppressive drug therapy,can be achieved.

Therapeutic methods of the invention provide the significant advantagesof allowing for delayed administration of immunosuppressive drugsfollowing transplantation, and at dosages below those administered inprior immunosuppressive drug therapies. Accordingly, the inventionavoids the need for administering high initial doses of broad-basedimmunosuppressive drugs that are currently used, and which are toxic tomost patients and/or which cause secondary diseases as a result ofextensive and extended immunosuppression.

Accordingly, in one embodiment, the invention provides a method forinducing tolerance to a transplant in a subject (e.g., a human) byadministering a therapeutically effective amount of an antagonist to afirst costimulatory molecule that is CD40, alone or in combination withan antagonist to a second costimulatory molecule, such as CD86. Theinitial dose of the antagonist is given before or at the time oftransplantation, followed by administration of a therapeuticallyeffective amount of an immuno-suppressive drug several days (e.g., atleast about 5 days up to 8 weeks) after transplantation. Multiple dosesof the antagonist and immunosuppressive drug are then continuouslyadministered sufficient to achieve long-term tolerance without hightoxicity to the subject.

The CD40 antagonist alone or in combination with the CD86 antagonist canbe administered to the subject using any suitable route ofadministration known in the art, such as injection or i.v. infusion, fora period of time sufficient to tolerize T cells to the transplant. Inparticular embodiments, the antagonist is administered over a period ofabout 6-12 weeks, or 12 weeks up to about 6 months, after the initialdose. In yet other embodiments, the antagonist is administered to thetransplant (e.g., organ or tissue) ex vivo prior to transplantation(e.g., by perfusion), followed by in vivo administration (to therecipient subject) after transplantation.

Suitable dosage regiments for the antagonist include those sufficient tomaintain inhibition of CD40 and CD86-mediated costimulation within thesubject until T cells are tolerized to the transplant. This can bejudged, for example, by the lack of any symptoms associated withrejection. For example, suitable dosages include those that achieveinitial and/or continuous serum levels of the antagonist of at leastabout 10-300 μg/ml, more preferably at least about 100-300 μg/ml, andmore preferably at least about 100-250 μg/ml. The dosages also can betapered during the treatment period. By tapered dosage or taperedadministration is understood administration of multiple doses indecreasing amounts, i.e. wherein an individual dose is equal to or lowerthan a preceding dose, and at least two individual doses are lower thantheir preceding ones.

As with the antagonist, the immunosuppressive drug can be administeredusing any suitable route of administration known in the art (e.g.,orally, by injection or i.v. infusion). Preferably, the first dose ofthe immunosuppressive drug is not administered until at least about 2,3, 4 or 5 days, more preferably at least about 1 week, more preferablyat least about 2 weeks, e.g. at least 3 weeks, 4 weeks, 6 weeks or even8 weeks ore more after transplantation, at which point T cells have beenfully or partially tolerized due to the antagonist treatment. In aparticular embodiment, the initial dose of the immunosuppressive drug isnot administered until completion of the administration (e.g., finaldose) of the antagonist (i.e., CD40 antagonist alone or in combinationwith a CD86 antagonist), but during a period where serum levels of theantagonist still remain. In another embodiment, the initial dose ofimmunosuppressive drug is delayed until the onset of transplantrejection, for example, upon appearance of at least one symptom ofrejection (e.g., in kidney transplantation, the rise of serum creatineand urea levels, as well as other rejection markers).

The immunosuppressive drug can be administered for a period of timeuntil tolerance to the transplant is achieved in the absence of theantagonist or the immuno-suppressive drug. For example, theimmunosuppressive drug can be administered over a period of about 5 daysto 26 weeks (6 months), e.g. 2-12 weeks or 4-8 weeks. Alternatively, theimmunosuppressive drug can be administered for longer periods ofapproximately 6-12 months, 12-24 months or longer. Preferably, thedosage of the immunosuppressive is tapered over the treatment period.For example, the initial dose of immunosuppressive drug can beadministered for a first period (e.g. 1-4 weeks) followed by a 50%reduction in the dose for a second period of e.g. 4-8 weeks, and afurther 50% reduction in the dose for a third period. Theimmunosuppressive drug initially can be administered at dosagesroutinely used in the clinic, or preferably even lower dosages that arestill sufficient to maintain tolerance and prevent graft rejection, andthen tapered over the course of time. For example, CsA can beadministered at a dose sufficient to achieve an initial serumconcentration level of about 300-500 ng/ml, followed by a serumconcentration level of about 200 ng/ml, followed by a serumconcentration level of about 100 ng/ml.

Suitable CD40 antagonists and CD86 antagonists that can be employed inthe methods of the invention include those that interfere with theability of these molecules to bind to their co-receptor (e.g., CD154 andCD28, respectively) and which inhibit CD40 and CD86-mediatedcostimulation, e.g., as measured by cytokine production and/or T cellproliferation. Exemplary antagonists include blocking antibodies andbispecific antibodies, soluble fusion polypeptides (e.g., CD86-Ig and/orCD40-Ig fusions and CD154-Ig and/or CD28-Ig fusions), peptides,peptidomimetics, nucleic acids, small molecules and the like.

In a particular embodiment, the antagonist is an antibody against CD40,CD86 and/or their respective co-receptors. Suitable antibodies can bederived from any species (e.g., human, murine, rabbit, etc.) and/or canbe engineered and expressed recombinantly (e.g., chimeric, humanized andhuman antibodies). The antibodies can be whole antibodies orantigen-binding fragments thereof including, for example, Fab, F(ab′)₂,Fv and single chain Fv fragments. The antibodies can also includeantagonistic bi-specific antibodies that bind to both CD40 and CD86, orto CD40 or CD86 and a second target molecule. In a particularembodiment, the CD40 antagonist is the chimeric anti-CD40 antibody,ch5D12, or a functionally equivalent antibody. In another particularembodiment, the CD86 antagonist is the chimeric anti-CD86 antibody,chFun-1, or a functionally equivalent antibody.

Suitable immunosuppressive drugs for use in the present inventioninclude those known in the art that are currently used for clinicalimmunosuppression following transplantation. These include, for example,signal 1 blockers, steroids and other drugs. Exemplary immunosuppressivedrugs include, but are not limited to, cyclosporine (CsA), tacrolimus(FK506), azathioprine, corticosteroids (e.g., prednisone), mycophenolatemofetil (MMF), rapamycin, anti-CD3 antibodies (e.g., OKT3), anti-CD25antibodies, and rapamycin. Combinations of two or more immunosuppressivedrugs also can be used. In a particular embodiment, theimmunosuppressive drug is a signal-1 blocker, e.g., cyclosporine, FK506,rapamycin and MMF.

The therapeutic method of the invention can be used to induce toleranceto a wide variety of transplanted tissues and organs. Accordingly, themethod can be used for broad-based treatment and/or prevention oftransplant rejection. Exemplary transplants (i.e., grafts) includeallografts, autografts, isografts and xenografts of organs (e.g.,kidney, liver, heart and lung), tissues (e.g., bone, skeletal matrix,skin) and cells (e.g., bone marrow, stem cells).

Other features and advantages of the instant invention will be madeapparent from the following detailed description and examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are expressly incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Kaplan Meyer plot of the time to rejection (as measured bythe first day serum creatinine is significantly increased) of allanimals in groups 1 and 2. ▴ indicate the animals treated with thecombination of ch5D12 and chFun-1 (group 2). ● indicate the animals witha low level of ch5D12 (group 1a). ▪ indicate the animals with a highlevel of ch5D12 (group 1b).

FIG. 2 is a Kaplan Meyer plot of the time to rejection (as measured bythe first day serum creatinine is significantly increased) of allanimals in groups 2 and 3. ▴ indicate the animals treated with thecombination of ch5D12 and chFun-1 (group 2). □ indicate the animalstreated with ATG and the combination of ch5D12 and chFun-1 (group 3).

FIG. 3 is a Kaplan Meyer plot of the time to rejection (as measured bythe first day serum creatinine is significantly increased) of allanimals in groups 3 and 4. ⋄ indicates untreated animals. □ indicateanimals treated with ch5D12 and chFun-1 pretreated with ATG (group 3). *indicate animals treated with ch5D12 and ch-Fun-1 followed by CsA (group4).

FIG. 4 is a bar graph depicting the incidence of rejection seen in day21 (FIG. 4A)) and day 42 (FIG. 4B) biopsies treated with (a) ch5D12 andchFun-1; (b) high dose ch5D12; or (c) CsA for 35 days (day 35 biopsiesin both panels). FIGS. 4C and 4D are bar graphs depicting the same dataexpressed as the mean biopsy scores for each group.

FIG. 5 is a graphic representation of CD40 expression analyzed using ananti-CD40 antibody that did not compete with 5D12 for binding to CD40and using 5D12/FITC.

FIG. 6 is a graphic representation of CD86 expression analyzed using ananti-CD86 antibody that did not compete with 5D12 for binding to CD86and using Fun-1/FITC.

FIG. 7 is a graphic representation showing the levels of CD8+ and CD4+ Tcells following transplantation in animals (group 3) treated withanti-CD40+anti-CD86 (high dose) and ATG.

FIG. 8 is a bar graph showing latent TGF-β development after treatmentwith anti-CD40, anti-CD40/CD86, and anti-CD40/CD86+Cyclosporin A.

DETAILED DESCRIPTION OF THE INVENTION

Therapeutic methods of the present invention have been shown tosuccessfully induce immune tolerance and long-term survival in a primatemodel of allograft transplantation for periods not previously observedin primate transplantation models (e.g., >700 days). Moreover, long-termsurvival did not require the continuous administration ofimmunosuppressive drugs, as is used in current transplantationtherapies. Accordingly, the invention provides methods for transplanttherapy that provide the significant advantages of long-term transplanttolerance, without causing the toxic side effects and secondary diseasesassociated with current transplantation therapies.

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined below, and additional definitions areset forth throughout the Detailed Description.

As used herein, the terms “CD40” and “CD86” refer to CD40 and CD86costimulatory molecules expressed on activated antigen presenting cells(see, for example, CD86 (B7-2) (Freeman et al. 1993 Science. 262:909 orGenBank Accession numbers P42081 or A48754); CD40 (Stamenkovic et al.EMBO 8:1403-1410, 1989 or GenBank Accession numbers CAA43045 andX60592.1), as well as fragments of CD40 and CD86 molecules, and/orfunctional equivalents thereof. The term “equivalent” is intended toinclude polypeptide sequences that have an activity of naturallyoccurring CD40 or CD86 molecules, e.g., the ability to bind CD40L orCD28, respectively, and modulate T cell costimulation.

As used herein, the term “CD40 antagonist” refers to agents (e.g.binding proteins, peptides and small molecules) that either inhibitfunctional responses mediated through CD40 signaling, or block and/orinhibit interaction of CD40 with CD40L (CD154). As used herein, the term“CD86 antagonist” refers to agents (e.g. binding proteins, peptides andsmall molecules) that either inhibit functional responses mediatedthrough CD86 interaction with CD28 and/or CTLA-4 (CD152), or blockand/or inhibit interaction of CD86 with CD28 and/or CTLA-4 (CD152). CD40and CD86 antagonists also block or inhibit CD40 or CD86-mediated T cellcostimulation. By blocking or inhibiting costimulatory signals, CD40 andCD86 antagonists are capable of preventing the activation of T cells andantigen presenting cells (e.g., cytokine production and T cellproliferation), thus inducing T cell anergy. A number of art recognizedreadouts of cell activation can be employed to measure the inhibition ofT cell costimulation, such as cytokine production or T cellproliferation assays, in the presence of CD40 and/or CD86 antagonists.

As used herein, the term “immunosuppressive drug” refers to drugs (e.g.,proteins, peptides, small molecules and hormones) that down-regulate anunwanted cellular and/or humoral immune response in an individual.Several immunosuppressive drugs are well known in the art and arecurrently used in clinical therapy including, for example, signal 1blockers, such as cyclosporine (CsA), tacrolimus (FK506), azathioprine,corticosteroids (e.g., prednisone), mycophenolate mofetil (MMF) andrapamycin. The term “signal-1 blocker” refers to an immunosuppressivedrug that interferes with T-cell receptor mediated signaling. Incontrast, antagonists to CD40 and/or CD86, as well as antagonists toother costimulatory molecules, can be defined as “signal 2 blockers”.Other immunosuppressive drugs include, for example, hormones (e.g.,steroids) and antibodies, such as anti-CD3 antibodies (e.g., OKT3) andanti-CD25 antibodies.

As used herein, the term “immune response” includes T cell mediatedand/or B cell mediated immune responses. Exemplary immune responsesinclude T cell responses, e.g., cytokine production and cellularcytotoxicity. In addition, the term immune response includes immuneresponses that are indirectly effected by T cell activation, e.g.,antibody production (humoral responses) and activation of cytokineresponsive cells, e.g., macrophages. Immune cells involved in the immuneresponse include lymphocytes, such as B cells and T cells (CD4⁺, CD8⁺,Th1 and Th2 cells); antigen presenting cells (e.g., professional antigenpresenting cells such as B lymphocytes, monocytes, dendritic cells,Langerhans cells, and non-professional antigen presenting cells such askeratinocytes, endothelial cells, astrocytes, fibroblasts,oligodendrocytes); natural killer cells; myeloid cells, such asmonocytes, macrophages, eosinophils, mast cells, basophils, andgranulocytes.

As used herein, the term “anergy” or “tolerance” refers to insensitivityof T cells to T cell receptor-mediated stimulation. Such insensitivityis generally antigen-specific and persists after exposure to thetolerizing antigen has ceased. For example, anergy in T cells (asopposed to unresponsiveness) is characterized by lack of cytokineproduction, e.g., IL-2. T-cell anergy occurs when T cells are exposed toantigen and receive a first signal (a T cell receptor or CD-3 mediatedsignal) in the absence of a second signal (a costimulatory signal).Under these conditions, re-exposure of the cells to the same antigen(even if re-exposure occurs in the presence of a costimulatory molecule)results in failure to produce cytokines and, thus, failure toproliferate. Anergic T cells can, however, proliferate if cultured withcytokines (e.g., IL-2). For example, T cell anergy can also be observedby the lack of IL-2 production by T lymphocytes as measured by ELISA orby a proliferation assay using an indicator cell line. Alternatively, areporter gene construct can be used. For example, anergic T cells failto initiate IL-2 gene transcription induced by a heterologous promoterunder the control of the 5′ IL-2 gene enhancer or by a multimer of theAP1 sequence that can be found within the enhancer (Kang et al. 1992Science. 257:1134).

As used herein, the term “graft” or “transplant” refers to an organ,tissue, or cell that has been transplanted from one subject to adifferent subject, or transplanted within the same subject (e.g., to adifferent area within the subject). Organs such as liver, kidney, heartor lung, or other body parts, such as bone or skeletal matrix, tissue,such as skin, intestines, endocrine glands, or progenitor stem cells ofvarious types, are all examples of transplants. The graft or transplantcan be an allograft, autograft, isograft or xenograft. The term“allograft” refers to a graft between two genetically non-identicalmembers of a species. The term “autograft refers to a graft from onearea to another on a single individual. The term “isograft” or“syngraft” refers to a graft between two genetically identicalindividuals. The term “xenograft” refers to a graft between members ofdifferent species.

As used herein, the term “acute rejection” refers to onset of a primaryimmune response to a graft, generally within days or weeks, and up toabout 6 to 12 months, after transplantation. The immune response iscaused by T cell recognition of the transplanted tissue associated withe.g., prominent local cytokine production, widespread pro-inflammatoryactivation of vascular endothelia, intense leukocyte infiltration, anddevelopment of graft-reactive, cytolytic T cells (CTL) that hastraditionally been associated with the acute loss of graft function.“Hyperacute rejection” is a type of rejection that occurs very rapidly,resulting in necrosis of the transplanted tissue within minutes or a fewhours of contact, and is caused by reactivity of the donor cells withpre-existing antibody.

As used herein, the terms “chronic rejection” refers to indolent,progressive immune responses that often occur one or more years aftertransplantation. Chronic rejection usually manifests in vascularizedsolid organ allografts as obliterative arteriopathy or graft vasculardisease (GVD), infiltration of immunocytes, interstitial andtubularatrophy, graft arteriosclerosis, and a marked fibrosis. “Graftversus host reaction (GVH),” as used herein, refers to the pathologicconsequences of a response initiated by transplanted immunocompetent Tlymphocytes into an allogeneic, immunologically incompetent host. Thehost is unable to reject the grafted T cells and the transplanted Tlymphocytes attack the tissues of the recipient due to recognition ofrecipient's Ags on recipient's MHC molecules (not necessarily byrecipient's tissues).

As used herein, the phrase “long-term tolerance” refers tolerance (i.e.,absence of rejection) of a graft or transplant in a subject for anextensive period of time, such as one or more years, preferably severalyears, and more preferably life. Complete tolerance occurs whentolerance is achieved and immunosuppressive treatment is no longernecessary.

II. Antagonists to CD40 and Other Costimulatory Molecules

A variety of antagonists to CD40 and other costimulatory molecules, suchas CD86, are known in the art and can be employed in the therapeuticmethods of the present invention.

Cell-to-cell signal exchange during antigen presentation deeplyinfluences the profile and extent of the immune response. Together withthe TCR/MHC-mediated signal, accessory signals are provided to the Tcell by the antigen-presenting cell (APC), through specificreceptor-ligand interactions that represent indispensable costimulationfor T-cell activation and survival. The main costimulatory pathways arethe B7 family members and the CD40-CD154 receptor-ligand pair. B7-1 andB7-2 costimulate T-cells by binding to CD28. Their binding is preventedby the neoexpression of CTLA-4, a CD28 homologue that can deliver anegative signal. Another CD28-like molecule, called ICOS (induciblecostimulator), has been described and binds B7RP-1, a third member ofthe B7 family, but not B7-1 and B7-2. The CD40-CD154 interaction worksas a two way costimulatory system by triggering activation signals toboth T-cell and APCs. Its importance is highlighted by the discoverythat mutations of the CD154 gene are responsible for a severe humanimmunodeficiency. Thus, disruption of the natural costimulatoryinteraction has can be highly effective for prevention and treatment oftransplant rejection.

Accordingly, suitable antagonists for use in the invention include thosethat block or inhibit the interaction of CD40 with its respectiveco-receptors, CD40L. Suitable antagonists to other costimulatorymolecules include those that antagonize the interaction (i.e.,costimulation pathway) between CD86 and CD28; OX40L and OX40; LIGHT andLIGHT-L; 4-1BBL and 4-1BB (CD137); CD80 and CTLA-4 (CD152), ICOS-L andICOS, and SLAM-L and SLAM (see e.g., Am. J. Respir. Crit. Care Med.(2000) 162(4): 164-168; J. Nephrol. (2002), 15: 7-16).

Such antagonists can be identified by a number of art recognized APC-and/or T-cell function assays, such as those described herein (e.g., Tcell proliferation and/or effector function, antibody production,cytokine production, and phagocyctosis).

Agents that block CD86 and/or CD40, also can be derived using CD40 andCD86 nucleic acid or amino acid sequences. The nucleotide and amino acidsequences of these costimulatory molecules are known in the art and canbe found in the literature or on a database such as GenBank. See, forexample, CD86 (B7-2) (Freeman et al. 1993 Science. 262:909 or GenBankAccession numbers P42081 or A48754); CD40 (Stamenkovic et al. EMBO8:1403-1410, 1989 or GenBank Accession numbers CAA43045 and X60592.1).

A. Antagonistic Antibodies

In a particular embodiment, the invention employs antagonisticantibodies to inhibit CD40 and/or CD86 function. As used herein, theterm “antibody” includes whole antibodies or antigen-binding fragmentsthereof including, for example, Fab, F(ab′)₂, Fv and single chain Fvfragments. Suitable antibodies include any form of antibody, e.g.,murine, human, chimeric, or humanized and any type antibody isotype,such as IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, or IgEisotypes.

Antibodies which specifically bind CD40 or its respective co-receptor,CD40L, to prevent CD40/CD40L interaction (e.g., CD40/CD40L-mediatedsignaling), can be used as CD40 antagonists in the present invention.Antibodies against other costimulatory molecules as described above,such as CD86 or its respective co-receptor, CD28, also can be used inthe present invention. As used herein, “specific binding” refers toantibody binding to a predetermined antigen. Typically, the antibodybinds with a dissociation constant (K_(D)) of 10⁻⁷ M or less, and bindsto the predetermined antigen with a K_(D) that is at least two-fold lessthan its K_(D) for binding to a non-specific antigen (e.g., BSA, casein)other than the predetermined antigen or a closely-related antigen.

Several CD86 antibodies are well known (see, for example, U.S. Pat. No.5,869,050; Powers G. D., et al. (1994) Cell. Immunol. 153, 298-311;Freedman, A. S. et al. (1987) J. Immunol. 137:3260-3267; Freeman, G. J.et al. (1989) J. Immunol. 143: 2714-2722; Freeman, G. L. et al. (1991)J. Exp. Med. 174:625-631; Freeman, G. J. (1993) Science 262:909-911; WO96/40915), and are also commercially available, e.g. from R&D Systems(Minneapolis, Minn.) and Research Diagnostics (Flanders, NJ). In aparticular embodiment, the CD86 antibody used in the invention is Fun-1,or a functional equivalent thereof (Nozawa et al., J. Pathol.169(3):309-15, 1993; Engel et al., Blood 84(5):1402-7, 1994). SeveralCD40 antibodies are also well known and readily available (see, forexample, U.S. Pat. No. 5,677,165). In a particular embodiment, the CD40antibody used in the invention is 5D12, or functional equivalentsthereof (DeBoer et al. (1992) J. Immunol. Methods 152(1):15-23).

The heavy and light chain variable sequences for Fun-1 and 5D12 areknown, as are antagonistic bispecific antibodies comprising the bindingregions of both Fun-1 and 5D12 (see e.g., US 2002/0150559).

Alternatively, antagonistic CD86 and CD40 antibodies can be producedaccording to well known methods for antibody production. For example,antigenic peptides of CD40, CD86 or their respective ligand or receptor,which are useful for the generation of antibodies can be identified in avariety of manners well known in the art. For example, useful epitopescan be predicted by analyzing the sequence of the protein usingweb-based predictive algorithms (BIMAS & SYFPEITHI) to generatepotential antigenic peptides from which synthetic versions can be madeand tested for their capacity to generate CD40, CD86, CD40L or CD28specific antibodies. Preferably, the antibody binds specifically orsubstantially specifically to the CD40 or CD86 molecule, or to theirrespective ligand or receptor, thereby inhibiting interaction ofCD40/CD40L or CD86/CD28, respectively.

Antagonistic antibodies used in the present invention can be monoclonalor polyclonal. The terms “monoclonal antibodies” as used herein, refersto a population of antibody molecules that contain only one species ofan antigen binding site capable of immunoreacting with a particularepitope of an antigen, whereas the term “polyclonal antibodies” refersto a population of antibody molecules that contain multiple species ofantigen binding sites capable of interacting with a particular antigen.Techniques for generating monoclonal and polyclonal antibodies are wellknown in the art (See, e.g., Current Protocols in Immunology, Coligan etal., eds., John Wiley & Sons, http://www.does.org/masterli/cpi.html).

Recombinant antagonistic CD40 and CD86 antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions can be made using standard recombinant DNA techniques, and arealso within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Patent Publication WO87/02671; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT Application WO 86/01533; Cabilly et al. U.S. Pat. No.4,816,567; Cabilly et al. European Patent Application 125,023; Better etal. (1988) Science 240:1041-1043; Liu et al. (1987) PNAS 84:3439-3443;Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; U.S. Pat. No. 5,225,539 5,565,332,5,871,907, or 5,733,743; Jones et al. (1986) Nature 321:552-525;Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J.Immunol. 141:4053-4060.

Recombinant chimeric antibodies can be further humanized by replacingsequences of the Fv variable region which are not directly involved inantigen binding with equivalent sequences from human Fv variableregions. General reviews of humanized chimeric antibodies are providedby Morrison, S. L., 1985, Science 229:1202-1207 and by Oi et al., 1986,BioTechniques 4:214. Those methods include isolating, manipulating, andexpressing the nucleic acid sequences that encode all or part ofimmunoglobulin Fv variable regions from at least one of a heavy or lightchain. Sources of such nucleic acid are well known to those skilled inthe art. The recombinant DNA encoding the chimeric antibody, or fragmentthereof, can then be cloned into an appropriate expression vector.Suitable humanized antibodies can alternatively be produced by CDRsubstitution U.S. Pat. No. 5,225,539; Jones et al. 1986 Nature321:552-525; Verhoeyan et al. 1988 Science 239:1534; and Beidler et al.1988 J. Immunol. 141:4053-4060.

Fully human antibodies that bind to CD40, CD86 and/or their respectiveligand or receptor can also be employed in the invention, and canproduced using techniques that are known in the art. For example,transgenic mice can be made using standard methods, e.g., according toHogan, et al., “Manipulating the Mouse Embryo: A Laboratory Manual”,Cold Spring Harbor Laboratory, which is incorporated herein byreference, or are purchased commercially. Embryonic stem cells aremanipulated according to published procedures (Teratocarcinomas andembryonic stem cells: a practical approach, Robertson, E. J. ed., IRLPress, Washington, D.C., 1987; Zijlstra et al. (1989) Nature342:435-438; and Schwartzberg et al. (1989) Science 246:799-803, each ofwhich is incorporated herein by reference). For example, transgenic micecan be immunized using purified or recombinant CD40 or CD86 or a fusionprotein comprising at least an immunogenic portion of the extracellulardomain of CD40 or CD86. Antibody reactivity can be measured usingstandard methods. The term “recombinant human antibody,” as used herein,includes all human antibodies that are prepared, expressed, created orisolated by recombinant means. Such recombinant human antibodies havevariable and constant regions derived from human germlineimmuno-globulin sequences. In certain embodiments, however, suchrecombinant human antibodies can be subjected to in vitro mutagenesis(or, when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo.

Single chain antagonistic antibodies that bind to CD40, CD86 or theirrespective ligand or receptor also can be identified and isolated byscreening a combinatorial library of human immunoglobulin sequencesdisplayed on M13 bacteriophage (Winter et al. 1994 Annu. Rev. Immunol.1994 12:433; Hoogenboom et al., 1998, Immunotechnology 4:1). Forexample, CD40, CD86, CD40L or CD28 can be used to thereby isolateimmunoglobulin library members that bind a CD40, CD86, CD40L or CD28polypeptide. Kits for generating and screening phage display librariesare commercially available and standard methods may be employed togenerate the scFv (Helfrich et al. J. Immunol Methods 2000, 237: 131-45;Cardoso et al. Scand J. Immunol 2000. 51: 337-44). Alternatively,Ribosomal display can be used to replace bacteriophage as the displayplatform (see, e.g., Hanes et al. Nat. Biotechnol. 18:1287, 2000; Wilsonet al. Proc. Natl. Acad. Sci. USA 98:3750, 2001; OR Irving et al., J.Immunol. Methods. 248:31, 2001).

In yet another embodiment of the invention, bispecific or multispecificantibodies that bind to CD86 and CD40 or antigen-binding portionsthereof. Such bispecific antibodies are described, for example, in US2002/0150559, and can be generated, e.g., by linking one antibody orantigen-binding portion (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to a second antibody orantigen-binding portion. Bispecific and multispecific molecules of thepresent invention can be made using chemical techniques, “polydoma”techniques or recombinant DNA techniques. Bispecific and multispecificmolecules can also be single chain molecules or may comprise at leasttwo single chain molecules. Methods for preparing bi- and multispecificmolecules are described for example in D. M. Kranz et al. (1981) Proc.Natl. Acad. Sci. USA 78:5807 and U.S. Pat. Nos. 4,474,893; 5,260,203;5,534,254. 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;5,013,653; 5,258,498; and 5,482,858.

Also within the scope of the invention are chimeric and humanizedantibodies in which specific amino acids have been substituted, deletedor added. In particular, preferred humanized antibodies have amino acidsubstitutions in the framework region, such as to improve binding to theantigen. For example, in a humanized antibody having mouse CDRs, aminoacids located in the human framework region can be replaced with theamino acids located at the corresponding positions in the mouseantibody. Such substitutions are known to improve binding of humanizedantibodies to the antigen in some instances. Antibodies in which aminoacids have been added, deleted, or substituted are referred to herein asmodified antibodies or altered antibodies.

The term modified antibody is also intended to include antibodies, suchas monoclonal antibodies, chimeric antibodies, and humanized antibodieswhich have been modified by, e.g., deleting, adding, or substitutingportions of the antibody. For example, an antibody can be modified bydeleting the constant region and replacing it with a constant regionmeant to increase half-life, e.g., serum half-life, stability oraffinity of the antibody. Any modification is within the scope of theinvention so long as the bispecific and multispecific molecule has atleast one antigen binding region specific for an FcγR and triggers atleast one effector function.

B. Fusion Protein Antagonists

Another form of CD40 and/or CD86 antagonist that can be employed in themethods of the present invention is a soluble form of (e.g., a fusionprotein or chimeric protein) CD40, CD86, their respective co-receptors(i.e., CD40L and CD28), or fragments and variants thereof. As usedherein, a CD40 or CD86 “chimeric protein” or “fusion protein” comprisesa CD40 or CD86 polypeptide, fragment, or functional variant thereof,operatively linked to a non-CD40 or CD86 polypeptide. Within a CD40 orCD86 fusion protein the CD40 or CD86 polypeptide can correspond to allor a portion of a CD40 or CD86 protein. In a particular embodiment, aCD40 or CD86 fusion protein comprises at least one biologically activeportion of a CD40 or CD86 protein, e.g., the extracellular domain of aCD40 or CD86 protein which binds to co-receptor. Within the fusionprotein, the term “operatively linked” is intended to indicate that theCD40 or CD86 polypeptide and the non-CD40 or CD86 polypeptide are fusedin-frame to each other. The non-CD40 or CD86 polypeptide can be fused tothe N-terminus or C-terminus of the CD40 or CD86 polypeptide.

A CD40 or CD86 fusion protein can be produced by recombinant expressionof a nucleotide sequence encoding a first peptide having CD40 or CD86activity and a nucleotide sequence encoding second peptide according tostandard techniques (e.g., see Current Protocols in Molecular Biology,eds. Ausubel et al. John Wiley & Sons: 1992). Preferably, the firstpeptide consists of a portion of the CD40 or CD86 polypeptide (e.g., aportion after cleavage of the signal sequence) that is sufficient tomodulate an immune response. The second peptide can include animmunoglobulin constant region, for example, a human Cγ1 domain or Cγ4domain (e.g., the hinge, CH2 and CH3 regions of human IgCγ1, or humanIgCγ4 (see e.g., Capon et al. U.S. Pat. Nos. 5,116,964; 5,580,756;5,844,095); a GST peptide, or an influenza hemagglutinin epitope tag(HA) (e.g. Herrsher et al., Genes Dev. 9:3067-3082, 1995).

The resulting fusion protein may have altered CD40 or CD86 solubility,binding affinity, stability and/or valency (i.e., the number of bindingsites available per molecule) and may increase the efficiency of proteinpurification. Fusion proteins and peptides produced by recombinanttechniques can be secreted and isolated from a mixture of cells andmedium containing the protein or peptide. Alternatively, the protein orpeptide can be retained cytoplasmically and the cells harvested, lysedand the protein isolated. A cell culture typically includes host cells,media and other byproducts. Suitable media for cell culture are wellknown in the art. Protein and peptides can be isolated from cell culturemedia, host cells, or both using techniques known in the art forpurifying proteins and peptides. Techniques for transfecting host cellsand purifying fusion proteins and peptides are known in the art.

Particularly preferred CD40 or CD86 fusion proteins include theextracellular domain portion or variable region-like domain of a humanCD40 or CD86 coupled to an immunoglobulin constant region (e.g., the Fcregion). Such fusion proteins can be monovalent or bivalent as isrecognized in the art. The immunoglobulin constant region may containgenetic modifications which reduce or eliminate effector activityinherent in the immunoglobulin structure. For example, DNA encoding theextracellular portion of a CD40 or CD86 polypeptide can be joined to DNAencoding the hinge, CH2 and CH3 regions of human IgGγ1 and/or IgGγ4modified by site directed mutagenesis, e.g., as taught in WO 97/28267.

C. Peptide Antagonists

A number of useful antagonists can also be derived from CD40 or CD86polypeptide sequences and their co-receptors. An antagonist may, forinstance, be a functional variant of the naturally occurring protein(e.g., a soluble form of CD40, CD86 or their respective co-receptors), amimic or peptidomimetic that inhibits the activity of CD40 or CD86required for the immunosuppressive effect. Variants of the CD40 or CD86proteins which serve as antagonists can be generated by mutagenesis(e.g., amino acid substitution, amino acid insertion, or truncation ofthe CD40 or CD86 protein), and identified by screening combinatoriallibraries of mutants, such as truncation mutants, of a CD40 or CD86protein for the desired activity, (e.g. CD40 or CD86 proteinantagonist).

For example, a variegated library of CD40 or CD86 variants can begenerated by combinatorial mutagenesis at the nucleic acid level, forexample, by enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential CD40 or CD86 sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display) containing the set of CD40 or CD86 sequencestherein. Chemical synthesis of a degenerate gene sequence can also beperformed in an automatic DNA synthesizer, and the synthetic gene thenligated into an appropriate expression vector. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem.53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)Nucleic Acid Res. 11:477.

Soluble forms of CD40, CD86 or their co-receptors can serve asantagonists in the methods of the invention. Such forms can beengineered using art recognized methods, and can comprise or consist of,e.g., an extracellular domain of a CD40 or CD86 protein. In oneembodiment, the extracellular domain of the CD40 or CD86 polypeptidecomprises the mature form of a CD40 or CD86 polypeptide, but not thetransmembrane and cytoplasmic domains. A soluble form of CD40 or CD86polypeptide, or a receptor binding portion thereof, which is multivalentto the extent that it is sufficient to crosslink the receptor is alsoconsidered an antagonist.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of CD40 or CD86 proteins. Themost widely used techniques, which are amenable to high through-putanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify CD40 or CD86 variants (Arkin and Youvan (1992) Proc. Natl.Acad. Sci. USA 89:7811-7815; Delagrave et al. (1993) Protein Eng.6(3):327-331).

Once suitable peptide antagonists are identified, systematicsubstitution of one or more amino acids of either CD40 or CD86 aminoacid sequence, or a functional variant thereof, with a D-amino acid ofthe same type (e.g., D-lysine in place of L-lysine) can also be used togenerate a peptide agonist which has increased stability. In addition,constrained peptides comprising a CD40 or CD86 amino acid sequence, afunctional variant thereof, or a substantially identical sequencevariation can be generated by methods known in the art (Rizo andGierasch (1992) Annu. Rev. Biochem. 61:387, incorporated herein byreference); for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

Peptides that act as antagonists CD40/CD40L or CD86/CD28 interactionscan be produced recombinantly or direct chemical synthesis. Further,peptides may be produced as modified peptides, with non-peptide moietiesattached by covalent linkage to the N-terminus and/or C-terminus. Incertain preferred embodiments, either the carboxy-terminus or theamino-terminus, or both, are chemically modified. The most commonmodifications of the terminal amino and carboxyl groups are acetylationand amidation, respectively. Amino-terminal modifications such asacylation (e.g., acetylation) or alkylation (e.g., methylation) andcarboxy-terminal-modifications such as amidation, as well as otherterminal modifications, including cyclization, can be incorporated intovarious embodiments of the invention. Certain amino-terminal and/orcarboxy-terminal modifications and/or peptide extensions to the coresequence can provide advantageous physical, chemical, biochemical, andpharmacological properties, such as: enhanced stability, increasedpotency and/or efficacy, resistance to serum proteases, and desirablepharmacokinetic properties.

Another form of antagonist is a peptide analog or peptide mimetic of theCD40 or CD86 protein. Peptide analogs are commonly used in thepharmaceutical industry as non-peptide drugs with properties analogousto those of the template peptide. These types of non-peptide compoundare termed “peptide mimetics” or “peptidomimetics” (Fauchere, J. (1986)Adv. Drug Res. 15:29; Veber and Freidinger (1985) TINS p. 392; and Evanset al. (1987) J. Med. Chem. 30:1229, which are incorporated herein byreference) and are usually developed with the aid of computerizedmolecular modeling. Peptide mimetics that are structurally similar toCD40 or CD86 or functional variants thereof, can be used to produce anantagonistic effect. Generally, peptidomimetics are structurally similarto the paradigm polypeptide (CD40 or CD86) but have one or more peptidelinkages (—CO—NH—) optionally replaced by a linkage selected from thegroup consisting of: —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH— (cis andtrans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. This is accomplished by theskilled practitioner by methods known in the art which are furtherdescribed in the following references: Spatola, A. F. in “Chemistry andBiochemistry of Amino Acids, Peptides, and Proteins” Weinstein, B., ed.,Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March1983), Vol. 1, Issue 3, “Peptide Backbone Modifications” (generalreview); Morley, J. S. (1980) Trends Pharm. Sci. pp. 463-468 (generalreview); Hudson, D. et al. (1979) Int. J. Pept. Prot. Res. 14:177-185(—CH₂NH—, —CH₂CH₂—); Spatola, A. F. et al. (1986) Life Sci. 38:1243-1249(—CH₂—S); Hann, M. M. (1982) J. Chem. Soc. Perkin Trans. I. 307-314(—CH═CH—, cis and trans); Almquist, R. G. et al. (190) J. Med. Chem.23:1392-1398 (—COCH₂—); Jennings-White, C. et al. (1982) TetrahedronLett. 23:2533 (—COCH₂—); Szelke, M. et al., EP 45665 (1982) CA: 97:39405(1982) (—CH(OH)CH₂—); Holladay, M. W. et al. (1983) Tetrahedron Lett.(1983) 24:4401-4404 (—C(OH)CH₂—); and Hruby, V. J. (1982) Life Sci.(1982) 31:189-199 (—CH₂—S—); each of which is incorporated herein byreference.

D. Nucleic Acid Antagonists

Nucleic acid molecules can also be used as antagonists of CD40 or CD86activity. For example, isolated nucleic acid molecules that areantisense molecules can be used as modulating agents to inhibit CD40and/or CD86 expression. An “antisense” nucleic acid comprises anucleotide sequence which is complementary to a “sense” nucleic acidencoding a protein, e.g., complementary to the coding strand of adouble-stranded cDNA molecule or complementary to an mRNA sequence.Accordingly, an antisense nucleic acid can hydrogen bond to a sensenucleic acid. The antisense nucleic acid can be complementary to anentire CD40 or CD86 coding strand, or only to a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding CD86 orCD40. The term “coding region” refers to the region of the nucleotidesequence comprising codons which are translated into amino acidresidues. In another embodiment, the antisense nucleic acid molecule isantisense to a “noncoding region” of the coding strand of a nucleotidesequence encoding CD40 or CD86. The term “noncoding region” refers to 5′and 3′ sequences which flank the coding region that are not translatedinto amino acids (i.e., also referred to as 5′ and 3′ untranslatedregions).

Given the coding strand sequences encoding CD40 and CD86 disclosed inthe art, antisense nucleic acids can be designed according to the rulesof Watson and Crick base pairing. The antisense nucleic acid moleculecan be complementary to the entire coding region of CD40 or CD86 mRNA,but preferably is an oligonucleotide which is antisense to only aportion of the coding or noncoding region of CD40 or CD86 mRNA. Forexample, the antisense oligonucleotide can be complementary to theregion surrounding the translation start site of CD40 or CD86 mRNA. Anantisense oligo-nucleotide can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acidfor use in the methods of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid molecule (e.g.,an antisense oligonucleotide) can be chemically or recombinantlysynthesized using naturally occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleic acids, e.g., phosphorothioatederivatives and acridine substituted nucleotides can be used.

Alternatively, an antisense nucleic acid molecule can be an a-anomericnucleic acid molecule, or a ribozyme. An a-anomeric nucleic acidmolecule forms specific double-stranded hybrids with complementary RNAin which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).Ribozymes are catalytic RNA molecules with ribonuclease activity whichare capable of cleaving a single-stranded nucleic acid molecule, such asan mRNA, to which they have a complementary region and can be used tocatalytically cleave CD40 or CD86 mRNA. Such molecules can beconstructed by methods known in the art. (see, e.g., Cech et al. U.S.Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742).

In another embodiment, CD40 or CD86 gene expression can be inhibited bytargeting nucleotide sequences complementary to the regulatory region ofthe CD40 or CD86 (e.g., the CD40 or CD86 promoter and/or enhancers) toform triple helical structures that prevent transcription of the CD40 orCD86 gene in target cells. See generally, Helene, C. (1991) AnticancerDrug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci.660:27-36; and Maher, L. J. (1992) Bioessays 14(12):807-15.

In another embodiment, a compound that promotes RNAi can be used toinhibit CD40 or CD86 expression. RNA interference (RNAi is apost-transcriptional, targeted gene-silencing technique that usesdouble-stranded RNA (dsRNA) to degrade messenger RNA (mRNA) containingthe same sequence as the dsRNA (Sharp, P. A. and Zamore, P. D. 287,2431-2432 (2000); Zamore, P. D., et al. Cell 101, 25-33 (2000). Tuschl,T. et al. Genes Dev. 13, 3191-3197 (1999)). The process occurs when anendogenous ribonuclease cleaves the longer dsRNA into shorter, 21- or22-nucleotide-long RNAs, termed small interfering RNAs or siRNAs. Thesmaller RNA segments then mediate the degradation of the target mRNA.Kits for synthesis of RNAi are commercially available from, e.g. NewEngland Biolabs and Ambion. In one embodiment one or more of thechemistries described above for use in antisense RNA can be employed.

In yet another embodiment, the CD40 or CD86 nucleic acid molecules ofthe present invention can be modified at the base moiety, sugar moiety,or phosphate backbone to improve, e.g., the stability, hybridization, orsolubility of the molecule. For example, the deoxyribose phosphatebackbone of the nucleic acid molecules can be modified to generatepeptide nucleic acids (see Hyrup, B. and Nielsen, P. E. (1996) Bioorg.Med. Chem. 4(1):5-23). As used herein, the terms “peptide nucleic acids”or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which thedeoxyribose phosphate backbone is replaced by a pseudopeptide backboneand only the four natural nucleobases are retained. The neutral backboneof PNAs has been shown to allow for specific hybridization to DNA andRNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup and Nielsen (1996) supra andPerry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670-675.

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652;WO88/09810) or the blood-brain barrier (see, e.g. WO89/10134). Inaddition, oligo-nucleotides can be modified with hybridization-triggeredcleavage agents (see, e.g., Krol et al. (1988) Biotechniques 6:958-976)or intercalating agents (see, e.g., Zon (1988) Pharm. Res. 5:539-549).To this end, the oligonucleotide can be conjugated to another molecule,(e.g., a peptide, hybridization triggered cross-linking agent, transportagent, or hybridization-triggered cleavage agent).

III. Immunosuppressants

Immunosuppressive drugs suitable for use in the present inventioninclude agents that down-regulate and/or suppress an immune response ina subject, for example, by blocking or inhibiting the activation orproliferation of T cells. Such drugs are well known in the art and arereadily available through commercial sources (see e.g., Immunobiology,Vol. 5 (chapter 14), ©2001 Garland Publishing, New York, N.Y., thecontents of which are incorporated by reference herein). In addition,such drugs are routinely used in current clinical therapies and, assuch, are easily adaptable to the methods of the present invention.

Transplant rejection is caused by detrimental immune responses againsttissue antigens. Thus, the goal of immunosuppressive drug therapy is todown-regulate such immune responses to avoid damage to the tissues ordisruption of their function. In the methods of the present invention,this is used in combination with therapies that induce T cell anergy ortolerance (e.g., anti-CD40 therapy alone or in combination withanti-CD86 therapy) to the tissues. This achieves effective, long-termprevention of transplant rejection.

Accordingly, a wide variety of known immunosuppressive drugs can be usedin the methods of the invention. Drugs currently used in the clinic tosuppress the immune system can be divided into three categories. First,anti-inflammatory drugs of the corticosteroid family, such asprednisone, are used. Second, cytotoxic drugs, such as azathioprine andcyclophosphamide, are used. Third, fungal and bacterial derivatives,such as cyclosporin A (CsA), FK506 (tacrolimus), and rapamycin(sirolimus), which inhibit signaling events within T lymphocytes, areused. Specifically, these fungal and bacterial derivatives exert theirbiological effects by binding to intracellular immunophilins, formingcomplexes that interfere with signaling pathways important for theclonal expansion of T lymphocytes.

The foregoing immunosuppressive drugs are all very broad in theiractions and inhibit protective functions of the immune system as well asharmful ones. Thus, it is well known that opportunistic infection is acommon complication of immuno-suppressive drug therapy.

In a particular embodiment, the immunosuppressive drug used in themethods of the present invention is a signal 1 blocker, such ascyclosporine (CsA), FK506, azathioprine, a corticosteroid, mycophenolatemofetil (MMF) and/or rapamycin. In other embodiments, theimmunosuppressive drug is a hormone (e.g., a steroid) or an antibody,such as anti-CD3 antibodies (e.g., OKT3) and anti-CD25 antibodies.

IV. Therapeutic Compositions

CD40 and/or CD86 antagonists can be formulated, separately or together,with a variety of pharmaceutically acceptable carriers prior toadministration. Similarly, immunosuppressive drugs can be formulatedwith a variety of pharmaceutically acceptable carriers prior toadministration. As used herein, “pharmaceutically acceptable carriers”include any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike that are physiologically compatible. Preferably, the carrier issuitable for intravenous, intramuscular, subcutaneous, parenteral,spinal or epidermal administration (e.g., by injection or infusion).

Suitable pharmaceutically acceptable carriers include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersion, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.In many cases, it will be preferable to include isotonic agents, forexample, sugars, polyalcohols such as mannitol, sorbitol, or sodiumchloride in the composition. Prolonged absorption of the injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, mono-stearate salts andgelatin. Supplementary active compounds can also be incorporated intothe compositions. Carriers that will protect the compound against rapidrelease, such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M., et al.(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids,hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonicacids and the like. Base addition salts include those derived fromalkali and alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methyl-glucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

The pharmaceutical compositions used in the methods of the presentinvention can be administered by a variety of methods known in the art.As will be appreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. Forexample, pharmaceutical formulations of the present invention includethose suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal and/or parenteral administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods known in the art of pharmacy. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will vary depending upon the subject beingtreated, and the particular mode of administration. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compositionwhich produces a therapeutic effect. Generally, out of one hundredpercent, this amount will range from about 0.01 percent to aboutninety-nine percent of active ingredient, preferably from about 0.1percent to about 70 percent, most preferably from about 1 percent toabout 30 percent.

Formulations of the present invention that are suitable for injectionmust be sterile and fluid to the extent that the composition isdeliverable by syringe. Proper fluidity can be maintained, for example,by use of coating such as lecithin, by maintenance of required particlesize in the case of dispersion and by use of surfactants. In many cases,it is preferable to include isotonic agents, for example, sugars,polyalcohols such as mannitol or sorbitol, and sodium chloride in thecomposition. Long-term absorption of the injectable composition can bebrought about by including an agent which delays absorption, forexample, aluminum monostearate or gelatin. Sterile injectable solutionscan be prepared by incorporating the active compound in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by sterilizationmicrofiltration. In the case of sterile powders for the preparation ofinjectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying (lyophilization) that yield a powder of theactive ingredient.

Formulations of the present invention which are suitable for the topicalor transdermal administration of compositions of this invention includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. Dosage forms for vaginal administration alsoinclude pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing such carriers as are known in the art to beappropriate. The active compound may be mixed under sterile conditionswith a pharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required. Alternatively, when theactive compound is suitably protected, as described above, the compoundmay be orally administered, for example, with an inert diluent or anassimilable edible carrier.

The pharmaceutical compositions of the invention may also containadjuvants such as preservatives, wetting agents, emulsifying agents anddispersing agents. Prevention of presence of microorganisms may beensured both by sterilization procedures, supra, and by the inclusion ofvarious antibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

V. Therapeutic Dosage Regimens

A variety of therapeutic dosage regimens can be employed in the methodsof the present invention according to the guidelines described below. Inall cases, the regimen involves administering to a subject, prior to orat the time of transplantation, a therapeutically effective amount of anantagonist of CD40 alone or in combination with and an antagonist ofCD86, followed (for example, at least several days later) byadministration of a therapeutically effective amount of animmunosuppressive agent(s).

The term “therapeutically effective” amount, as used herein, refers to adosage of antagonist or immunosuppressive drug that induces complete orsubstantial tolerance to a transplant in a subject (e.g., 80% tolerancerelative to untreated subjects). Lack of tolerance, i.e., rejection, canbe measured by the development of one or more symptoms associated withgraft rejection including, but not limited to, a substantial rise inserum creatine levels, reduced organ function, pain or swelling in thelocation of the organ or tissue, fever, and/or general discomfort.Conditions associated with graft rejection include, without limitation,acute graft rejection, graft-versus-host disease (GVHD), chronic graftrejection (e.g., chronic/sclerosing nephropathy).

Accordingly, tolerance (e.g., the prevention and/or reduction in signsand/or symptoms of acute and/or chronic transplant rejection), can beevaluated using art-recognized assays and methods known to measure theaforementioned symptoms of transplant rejection. These include, e.g.,the assays and parameters described in the Examples provided below, suchas those that measure serum creatine levels and donor-specific antibodylevels, as well as needle biopsies etc.). Alternatively, tolerance canbe evaluated by examining the reduction of T cell activation andproliferation using standard assays. One of ordinary skill in the artalso can determine such therapeutically effective amounts based onfactors such as the subject's size, the severity of the signs and/orsubject's symptoms, and the particular composition or route ofadministration selected.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

Accordingly, dosage regimens are adjusted to provide the optimum desiredresponse in a subject that is sufficient to maintain the blockage orinhibition of CD40/CD40L interaction, or CD40/CD40L interaction andinteraction of other costimulatory molecules, such as CD86/CD28, untilgraft tolerance is induced. For example, the dosage regimen can beadjusted to achieve sufficient serum levels of antagonist to achievefull coating of substantially all CD40 and/or CD86 molecules expressedand/or to inhibit or block the functional activity of substantially allCD40 and/or CD86 molecules expressed within the transplant subject. Whenadministering both CD40 and CD86 antagonists, the antagonists can beco-administered simultaneously in the same pharmaceutically acceptableexcipient, or co-administered one after the other in separatepharmaceutically acceptable excipients.

The initial administration of the CD40 and CD86 antagonists can bebefore transplantation (e.g., about 1 week, or 5, 4, 3, 2 or 1 day(s)prior to transplantation), at the time of transplantation (e.g., on day0), or shortly following transplantation (e.g., 1 or 2 days aftertransplantation), with repeated dosages thereafter for a sufficientperiod to substantially tolerize T cells to the transplant. Theantagonis(s) can also be administered in multiple doses (e.g., daily,every 2 days, every 3 days, once weekly or once bi-weekly, orcombinations thereof, over a period of time lasting, for example,approximately 2-26 weeks, 4-16 weeks, 6-12 weeks or 8-10 weeks after theinitial dose.

In particular embodiments, the initial dosage of the antagonist(s) isapproximately 1 to 20 mg/kg, 1 to 10 mg/kg, or 1 to 5 mg/kg, withsubsequent doses being reduced to approximately 0.1 to 10 mg/kg or 1 to5 mg/kg. In other particular embodiments, the dosages used aresufficient to maintain an initial or continuous serum level of theantagonist(s) of at least about 10-300 μg/ml, 75-250 μg/ml, 100-250μg/ml, 150-250 μg/ml or 100-200 μg/ml during the treatment period.

Initial dosages of the antagonist can also be administered ex vivo tothe transplant prior to transplantation into the subject, followed by invivo administration thereafter. For example, the transplant can bewashed and/or perfused using well-known methods in media containing theantagonist in an amount sufficient to sufficient to saturatesubstantially all CD40 and/or CD86 molecules, and/or their respectiveligand molecules, in the donor tissue. The transplant can then besurgically grafted into the recipient subject. The term “saturate”refers to binding to CD40 and/or CD86 molecules, and/or their respectiveligand molecules, resulting in a functional antagonistic effect on themolecule. This includes, but is not limited to, inhibiting or blockingthe interaction of the molecules with their respective ligands.

As with the CD40 and CD86 antagonists, the dosage regimens of theimmunosuppressive drug(s) can also be adjusted to provide the optimumtherapeutic benefit (e.g., long-term survival and tolerance). In allcases, the optimum dosage regimen includes the lowest amount of drugnecessary to maintain anergy to the transplant following or duringtreatment with antagonist. This preserves the health of the transplantfrom toxicity and the overall strength of the recipient's immune system.In addition, the toxic effects associated with early immunosuppressivedrug treatment (e.g., organ toxicity and/or systemic toxicity) areavoided by delaying administration of the immunosuppressive drug untilthe antagonist treatment regimen is completed or nearly completed. Forexample, the immunosuppressive drug(s) can be administered at leastseveral days after transplantation, e.g., at least about 2, 3, 4 or 5days, preferably at least about 1 week, more preferably at least about2-8 weeks, and more preferably at least about 6-8 weeks aftertransplantation.

In a particular embodiment, to maximize the time before administeringthe immunosuppressive drug, the initial dose of the immunosuppressivedrug is delayed until the final dose of the antagonist(s) (i.e., CD40antagonist alone or in combination with a CD86 antagonist) has beenadministered. Alternatively, the immunosuppressive drug can be delayeduntil the first symptoms of acute or chronic rejection are observed,which can be prior to the final administration of the antagonists, orcan be after the final administration of the antagonists, depending onthe subject being treated. Accordingly, in cases where the subject doesnot demonstrate early signs of graft rejection (e.g., due to toleranceinduced by the initial CD40/CD86 antagonist therapy), treatment with theimmunosuppressive drug can be delayed as long as possible aftertransplantation, e.g., at least 6 to 8 weeks, in order to minimize thedoses required to maintain or establish tolerance and to reducetoxicity.

The immunosuppressive drug can be administered in multiple doses (e.g.,daily, every 2 days, every 3 days, once weekly, once bi-weekly ormonthly) over a period of time lasting until the patient is tolerized.In certain subjects, this can be achieved in as little as 2-12 weeks,although longer periods (e.g., up to six months, one year, two years orlonger after the initial dose of the drug) are required to maintaintolerance in other patients. Notwithstanding, due to the initialtolerance achieved by the CD40 and/or CD86 antagonist, the dosage levelsof immunosuppressive drug required to maintain tolerance are generallyexpected to be significantly lower than those used in current clinicaltransplantation therapy.

For example, when using CsA, the immunosuppressive drug can beadministered at a dosage of up to about 10 mg/kg or 1 to 5 mg/kg for aperiod of time of about 1 week, 2 weeks, 3 weeks or up to about 1 month,with subsequent doses being reduced to approximately 0.1 to 10 mg/kg or1 to 5 mg/kg for the remaining treatment period. In other embodiments,CsA can be administered at a dose of about 5 to 10 mg/kg for a timeperiod of 1 to 4 weeks, followed by a 50% reduction in the dose for asecond time period of 1 to 4 weeks, and a further 50% reduction in thedose for a third period of time of 1 to 4 weeks, or up to 6 months. Inother particular embodiments, CsA can be administered at dosagessufficient to achieve an initial serum concentration level of about300-500 ng/ml for 4 about weeks, about 200 ng/ml for the following 4weeks, and about 100 ng/ml for an additional 4 weeks.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting.

EXAMPLES

The following studies were performed to study the efficacy of CD40 andCD86 antagonist therapy to induce long-term immune tolerance in a rhesusmonkey kidney allograft model, without the need for initial or highdoses of immunosuppressive agents currently used. Primates were chosenfor this study because the results obtained using this animal model areknown to correlate with clinical transplantation more closely than thoseobserved in inbred murine disease models (since non-human primates, likehumans, are outbred species). In addition, monoclonal antibodies (Mabs)raised against human costimulatory molecules are cross-reactive in mostnon-human primate species, providing a more direct analysis ofclinically relevant agents.

Specifically, treatment regimens were designed to inhibit the onset of Tcell co-stimulation by blocking CD40 signaling or CD40 and CD86signaling, while still permitting CD80 to interact with CTLA4 (CD152)thus maintaining the signals required for T-cell down-regulation. Thistreatment, which allows for the induction of immune tolerance to thegraft, is then followed by treatment with immunosuppressive drugs tomaintain inhibition of T-cell responses until tolerance is established.

The results obtained from these experiments demonstrate that delayingtreatment with immunosuppressive drugs not only results in reducedtoxicity, but unexpectedly, promotes long-term survival and grafttolerance, even after immunosuppressive therapy is terminated. Thus, thetherapeutic approaches described herein provide the substantialadvantage of avoiding the risk of serious infections and cancerassociated in current daily clinical practice using life-longimmunosuppressive therapies.

Materials and Methods

Animals

Naive, captive bred 4-6 kg rhesus monkeys (Macaca mulatta) were eitherborn and raised at the Biomedical Primate Research Center (TheNetherlands) (BPRC) or were purchased from a commercial breedingstation. The animals were fed monkey chow supplemented by fresh fruitand vegetables, and tap water was provided ad libitum. All procedureswere performed in accordance with guidelines of the Animal Care and UseCommittee installed by Dutch law. All animals used in the study were inoverall good health; had normal hematology and clinical chemistryvalues; had no history of allo-immunization; had, no history ofimmunization with human or murine serum component; and were MHC and ABOtyped.

All animals were typed for Mamu-A, B and DR antigens by serology(Bontrop et al. 1995). Disparity for DR locus antigens was confirmed byDRB typing (Doxiadis et al. 2000). Recipients were mismatched for one ortwo Mamu-DR antigens, and had at least one Mamu-A and -B mismatchedantigen with the donor. Total Mamu-DR mismatches were distributedequally in both groups. The recipient-donor pairs were compatible forABO-antigens (Doxiadis et al. 1998). In addition, the stimulation indexof the one-way mixed lymphocyte reaction of the recipient cells directedagainst the donor antigens was positive (SI>3). All animals werescreened for pre-existing antibodies to ch5D12 and chFun-1 by ELISA (seeAnti-Chimeric-antibody responses (RACA)).

Production and Purification of Chimeric Anti-Human Anti-CD40 andAnti-CD86

NS0 cells were transfected simultaneously by electroporation with thetwo expression plasmids encoding for the variable light and variableheavy chain of the anti-CD40 Mab (ch5D12) and anti-CD86 (chFun-1) Mab,respectively. For both Mabs, stable cell-lines were selected using G418and mycophenolic acid as selection markers. Cell-lines were thenscreened for high production levels. A high producing cell-line for eachMab was then expanded by growth in a shaker flask and adapted toserum-free production medium. After a last quality check for each Mab,large amounts of material were obtained by growth in a bioreactor, andthe Mabs were purified using protein A followed by gel-filtration.

Purified protein concentrations of both Mabs the protein was determinedaccording to standard methods. Binding to CD86 or CD40 was then testedby ELISA, and by FACS on B cells. These analyses demonstrated equivalentbinding of the chimeric Mabs compared to their respective mousecounterparts. Each of the chimeric Mab preparations was then furthertested for purity and endotoxin levels and was found to meet thestandards for in vivo use in non-human primates.

Kidney Transplantation

Heterotopic kidney allo-transplantation with bilateral nephrectomy wasperformed as described previously (Neuhaus et al. 1982, Ossevoort et al.1999). The clinical condition of the animals was monitored by dailyvisual inspection and by frequent haematological and clinical chemistryblood values determined in a clinical laboratory (SSDZ, Delft) or at theBPRC. Needle biopsies (18G, BARD, The Netherlands) were taken from thekidney at 2, 6, 10, 16 and 26 weeks after transplantation. The biopsieswere stored in formaline and/or cryopreserved for later analysis.

Transplant rejection was monitored by increases in serum creatinine andurea levels (Haanstra et al. 2003). A rejection episode was not treated.When serum creatinine showed a significant rise or when the clinicalcondition began to deteriorate, the animals were euthanized and acomplete necropsy was performed in which the abdominal and thoraciccavities were opened and internal organs examined in situ and preservedin a neutral aqueous phosphate-buffered 4% solution of formaldehyde. Forhistological examination, biopsy material and tissues from the necropsywere formalin-fixed and paraplast-embedded, and samples of the spleenand graft were also cryopreserved.

Biopsies were analyzed by four-micron-thick sections were stained withhematoxylin and eosin (H&E), periodic acid Schiff, and a silverimpregnation stain (Jones) (Haanstra et al. 2003). Histomorphologicalevaluation of allograft rejection was performed according to the Banffclassification (Racusen et al. 1999).

Mab and Drug Treatment.

Group 1 animals (n=7) were treated with anti-CD40 alone. Two animals(Group 1a) received two initial doses of 10 mg/kg i.v. of the Mab on day−1 and day 0, followed by 5 mg/kg on days 4, 7, 11, and 14 and 5 mg/kgi.v. weekly thereafter until day 56. Circulating Mab levels in these twoanimals were found to be lower than 100 μg/ml serum after day 14.Therefore, the remaining animals in the first group (Group 1b) weretreated with a doubling of the dosing schedule, 20 mg/kg on days −1 and0, on days 4, 7, 11, and 14 with 10 mg/kg and with 5 mg/kg twice weeklythereafter until day 56. No additional immunosuppression or rescuemedication was provided to these animals.

Group 2 animals (n=6) were treated with a combination of anti-CD40 andanti-CD86. Two animals (Group 2a) received two initial doses of 10 mg/kgi.v. for each Mab on day −1 and day 0, followed by 5 mg/kg i.v. on days4, 7, 11, and 14 and 5 mg/kg bi-weekly thereafter until day 56. Again,circulating Mab levels were found to be lower than 100 μg/ml serum afterday 14, and therefore subsequent animals (Group 2b) were treated with adoubling of the dosing schedule, 20 mg/kg on days −1 and 0, on days 4,7, 11, and 14 with 10 mg/kg and with 5 mg/kg twice weekly thereafteruntil day 56. No additional immunosuppression or rescue medication wasprovided to these animals.

Group 3 animals were pretreated with 20 mg/kg Thymoglobuline (ATG)(Imtix-Sangstat) on day −1 (i.v.) and 10 mg/kg on day 0, followed by 10mg/kg anti-CD40+anti-CD86 on days 4, 7, 11 and 14, and 5 mg/kg bi-weeklythereafter until day 56 to a serum level of at least 250 μg/ml. Theanimals were further treated with CsA from day 42-100 onward with 5-10mg/kg i.m. In addition, the animals were treated on day −1 with 10 mg/kgSolumedrol (methylprednisolon, Pharmacia & Upjohn). After day 42 thefirst rejection episode in each animal was treated with 3 days of 10mg/kg Solumedrol, and animals continued on CsA (Novartis) and 1 mg/kgprednisolon, tapering after day 90 (Nourypharma) thereafter.

Group 4 animals were treated with a combination of 20 mg/kg ofanti-CD40+ anti-CD86 on days −1 and 0, 10/mg/kg of anti-CD40+anti-CD86on days 4, 7, 11 and 14, followed by 5 mg/kg bi-weekly for 8 weeks i.v.to a serum level of at least 250 μg/ml. CsA was administered from days42-124 orally twice weekly and i.m. five times weekly to obtain bloodconcentration levels of 300 ng/ml for 4 weeks, 200 ng/ml for thefollowing 4 weeks, and 100 ng/ml for the final four weeks.

Historical controls were treated with CsA 10 mg/kg i.m. daily forthirty-five days, with detectable serum levels until day 70. A summaryof the dosing schedules used is depicted in Table 1. TABLE 1 DosingSchedule EXPERIMENTAL GROUP TREATMENT Dosing 1a Anti CD40 alone day −1,0: 10 mg/kg; low dose day 4, 7, 11, 14, 21, 28, 35, 42, 49, 56: 5 mg/kg1b day −1, 0: 20 mg/kg; Anti CD40 alone day 4, 7, 11, 14: 10 mg/kg; highdose day 18, 21, 25, 28, 32, 35, 39, 42, 46, 49, 52, 56: 5 mg/kg 2a AntiCD40 + day −1, 0: 10 mg/kg; anti CD86 day 4, 7, 11, 14, 21, 28, 35, 42,49, 56: 5 mg/kg low dose 2b Anti CD40 + day −1, 0: 20 mg/kg; anti CD86day 4, 7, 11, 14: 10 mg/kg; high dose day 18, 21, 25, 28, 32, 35, 39,42, 46, 49, 52, 56: 5 mg/kg 3 Anti CD40 + day −1, 0: 20 mg/kg; anti CD86day 4, 7, 11, 14: 10 mg/kg; high dose day 18, 21, 25, 28, 32, 35, 39,42, 46, 49, 52, 56: 5 mg/kg thymoglobuline (ATG) day −1: 20 mg/kg i.v.;day 0: 20 mg/kg s.c. solumedrol day −1: 10 mg/kg and after day 42 incase of rejection 3 × 10 mg/kg cyclosporine day 42-100: 5-10 mg/kg i.m.di-adreson-F after day 42, subsequent to solumedrol treatment: 1 mg/kg,taper after day 90. 4 Anti CD40 + day −1, 0: 20 mg/kg; anti CD86 day 4,7, 11, 14: 10 mg/kg; high dose day 18, 21, 25, 28, 32, 35, 39, 42, 46,49, 52, 56: 5 mg/kg cyclosporine day 42-126: oral 2× weekly, IM 5×weekly target blood levels: 4 weeks 300 ng/ml; 4 weeks 200 ng/ml; 4weeks 100 ng/mlRhesus Anti-Chimeric Antibody (RACA) Titers

Blood samples (clotted blood) were collected at regular time points,pre- and post-transplantation from the femoral vein in the groin usingaseptic techniques: Vacutainer blood collection systems (BectonDickinson, Vacutainer systems, France) were used. Serum was collected bycentrifugation and stored at −80° C. until further use.

For the determination of rhesus-anti-chimeric antibody (RACA) IgGresponses against ch5D12 and chFun1, 96-well flat-bottom ELISA plateswere coated with 1 μg/ml murine 5D12 or murine Fun1. Plates wereincubated overnight at 4° C. or 1 hr at 37° C. with 100 ng/well and 500ng/well to determine the IgG RACA and the IgM RACA response,respectively. The plates were washed on an automated washer and blockedwith 200 μl 1% BSA (RIA grade) in PBS for 1 hr at 37° C. The plates thenwere emptied and incubated for 2 hrs at 37° C. with 100 μl/well ofserial dilutions of the serum samples. After washing, plates wereincubated with alkaline phosphatase-labeled rabbit-anti-monkey-IgG(Sigma, The Netherlands). The plates were then washed again, followed byaddition of 100 μl/well substrate (p-Nitrophenyl Phosphate (pNPP)).Absorbance was measured at 405 nm. The first dilution of IgG RACA to behigher than three times the pre-transplantation value was taken as theabsolute titer. IgM antibodies were expressed as index ofpost-transplantation values divided by pre-transplantation values,because pre-transplantation values had considerable background andinter-animal variation. The IgM RACA was considered to be positive whenthe index was 1.5 or higher on two or more consecutive time points.

Donor-Specific Antibodies

Blood samples (clotted blood) were collected at regular time points,pre- and post-transplantation from the femoral vein in the groin usingaseptic techniques: Vacutainer blood collection systems (BectonDickinson, Vacutainer systems, France) were used. Serum was collected bycentrifugation and stored at −80° C. until further use.

Anti-donor antibodies were assessed by incubating donor spleen cellswith recipient serum. Since circulating chimeric Mabs in the recipientserum bound to donor spleen cells, and this was detected by the rabbitanti-human IgG and IgM antibodies, donor spleen cells were pre-incubatedfor 30 min. at 4° C., with mouse anti-human 5D12 (CD40) and Fun-1 (CD86)Mabs provided by PanGenetics BV. Donor spleen cells were alsopre-incubated with 50 μl 1/20 diluted rabbit anti-human Ig (DAKO,Derunark) to block aspecific antibody binding. Cells were washed withFACS buffer (0.5% BSA, 0.05% NaN₃ in PBS). Cells were then incubatedwith 25 μl recipient serum, at 4° C. for 30 min. Cells were washed againand incubated with rabbit anti-human IgG- or IgM-FITC F(ab′)₂ (DAKO,Denmark, dilution 1/20). Cells were washed and fixated withformaldehyde. Before analysis cells were washed to remove theformaldehyde and resuspended in PBS. Cells were analyzed on a FACScan(BD, Mountain View, Calif., USA) using standard settings for lymphocyteanalysis.

Immunophenotyping—FACS Analysis

Subset analyses were performed at regular time points using whole bloodin EDTA. The blood was washed with FACS buffer (PBS/BSA/NaN₃) to removecirculating free Mab present in the serum. The samples were incubatedwith either fluorescein isothiocyanate (FITC)-labeled ch5D12 or chFun-1to detect in vivo coating of the cells, or with either anon-crossblocking FITC-labeled anti-CD40 Mab (clone 26, PanGenetics, BV)or phycoerthrin-labeled anti-CD86 Mab (IT2.2, Becton DickinsonPharMingen, San Diego, Calif.) to detect the percentage of positivecells for CD40 and CDD86. CD3, CD4, CD8 and CD20 positive populationswere also monitored, by using clones SP34 for CD3 (BD PharMingen, CA,USA) and clones SK3, SK1, and L27 for CD4, CD8 and CD20 respectively(BD, PharMingen). A negative control was also included. The cells wereincubated for 30 min. at 4° C. The red blood cells were lysed using FACSLysing Solution (BD, CA, USA), for 10 min. at room temperature. Cellswere washed 2 times and fixated using formaldehyde. Fluorescence wasmeasured within 48 hrs. Analysis was performed using CellQuest software(BD, CA, USA). Lymphocytes were analyzed for CD40 and CD86 coating invivo, and for CD40, CD80 and CD86 expression using CellQuest software(Becton Dickinson).

Example 1 Onset of Graft Rejection

The serum creatinine and urea levels of each animal were monitoredbecause they are the first parameters to rise when kidney function isimpaired, thus serving as an early indicator of graft rejection (e.g.,acute rejection). However, in the week immediately post transplantationserum creatinine and urea may also be elevated due to thetransplantation procedure. When the rise in serum creatinine and urea isdue to the transplant rejection, electrolytes also show abnormal values.

The results of this study are summarized in Table 2. The day at whichthe rejection process started was no different between groups 1a+b andgroups 2a+b. However, it should be noted that in group 1a+b, whichreceived anti-CD40 alone, some animals showed a short graft survival andothers did not reject until several months after Mab treatment wasstopped. Animals with a short graft survival which received a low doseof ch5D12 (BJG and 96087) did not show graft rejection in the kidney(C008, circulatory problem) or had a low level of circulating ch5D12(RI075). Thus, group 1 could be subdivided into short surviving animalstreated with a low level of ch5D12, and long surviving animals treatedwith a high level of ch5D12 (see FIG. 1). The median time to rejectionwas 28 days for group 1a, 126 days for group 1b and 70 days for group 2.This represents a statistically significant difference among thesegroups (Cox's proportional hazard analysis). TABLE 2 Day 4 creatinineDay first significant Day Group Animal (μmol/liter) rise serumcreatinine euthanized 1a BJG 279 4 8 1b C008 106 11 12 1a 96087 108 2830 1b* RI075 874 35 42 1b RI208 137 82 91 1b RI149 125 126 134 1b DXW114 175 217 2b RI140 564 53 61 2a EBP 103 70 71 2b RI055 318 39 75 2bC118 129 74 78 2b C128 179 113 119 3 RI251 114 38 38 3 RI286 410 21 56 3RI203 87 49 56 3 RI139 345 56 97 RI204 98 42 109 4 DKW 97 133 141 4RI279 81 222 231 4 RI301 85 >700 >700 4 97064 88 >700 >700Untreated control animals rejected their grafts within one week (n = 4)

A comparison of the time to rejection of the animals in groups 2 and 3,and groups 3 and 4 are shown in FIGS. 2 and 3, respectively. The mediantime to rejection was 42 days for group 3, a statistically significantdifference when compared to 70 days for group 2, indicating that theaddition of ATG could be detrimental to graft survival when administeredwith the combination of Mabs.

In contrast, as shown in FIG. 3, none of the animals in group 4, whichreceived anti-CD40+anti-CD86 followed by CsA showed a significant risein serum creatine levels during the treatment period indicating that themedian time to the onset of rejection was significantly longer in group4 than the time to rejection in all other groups. Moreover, in at least2 animals of group 4, serum levels remained within a normal range oncetreatment was terminated.

Example 2 Pathology of Chronic Graft Rejection

As discussed herein, chronic rejection due to continuous immuneactivation and subsequent tissue damage is the major problem in currenttransplant medicine. For this reason, kidney biopsy specimens were alsotaken at several time points (e.g., days 21, 42, 70) for the animals ingroups 1 and 2, and compared to control animals that were treated withCsA alone (10 mg/kg i.m. daily for 35 days).

As shown in Table 3 and FIG. 4, both infiltrate scores and tubulitisscores were reduced in animals treated with anti-CD40 oranti-CD40+anti-CD86 when compared to the CsA treated controls. Moreover,on biopsies from days 21 and 42, less interstitial infiltration ortubulitis was present in animals treated with the combination of Mabsthan in animals treated with ch5D12 alone. Thus, it seems that thecombination of Mabs prevented graft infiltration. However, it is alsopossible that the infiltrating cells seen in the animals treated withch5D12 alone were not necessarily pathogenic and may even have containedregulatory or suppressor T cells

Example 3 Graft Pathology after Euthanasia

Animals were euthanized before they became clinically ill due to therejection process, and pathology was performed to determine the extentof tissue rejection. A comparison of the Banff scores for each animal issummarized in Table 3.

Of the seven animals treated with anti-CD40 alone (group 1), threerejected the transplant while still on treatment. Two of these animalsreceived the lower dose of anti-CD40 (group 1a). One animal died after12 days due to a blocked ureter and had only borderline signs ofrejection, and the remaining three animals did not reject their graftduring treatment, but at variable times after cessation of treatment.

None of the animals treated with the combination of anti-CD40 andanti-CD86 showed signs of graft rejection during treatment (group 2).However all animals rejected the kidney transplant around the end of thetreatment period. Only one animal had only borderline signs of kidneyrejection, but had a blocked ureter (EBP), which could also have beencaused by a rejection process. The two animals that were euthanized onday 3 and 7, respectively, did not show signs of rejection. Theseanimals were excluded from the statistical analysis. Thus, whiletreatment with anti-CD40 appears to result in a variable delay of graftrejection, the combination of anti-CD40 and anti-CD86 appears tocompletely prevent graft rejection during treatment, providing a moreconsistent delay in graft rejection than anti-CD40 alone. TABLE 3 Daystart Day of Graft pathology at necropsy: GROUP TREATMENT ANIMALrejection euthanasia Acute rejection/CAN 1a anti CD40 Ld BJG 4 8 acuterejection I-II 96087 28 30 acute rejection III 1b anti CD40 Hd C008 1112 borderline rejection Ri075 35 42 acute rejection II/CAN I Ri208 82 91acute rejection II/CAN III Ri149 126 134 acute rejection II/CAN III DXW175 217 acute borderline/CAN (I or) III 2a anti CD40 + 96079 7 norejection, CMV 86 Ld EBP 70 71 acute rejection borderline to I 2b antiCD40 + C146 3 PNF, no rejection 86 Hd Ri140 53 61 acute rejectionII-III/CAN 0-I Ri055 39 75 acute rejection I C118 74 78 acute rejectionI-II/CAN C128 113 119 acute rejection 0-I 3 anti CD40 + Ri0271 8 PNF, norejection 86 Hd Ri0251 38 38 acute rejection III ATG, steroids Ri286 2156 acute rejection I CsA Ri203 49 56 no rejection Ri139 56 97 acuterejection I Ri204 42 109 acute rejection I/CAN II-III 4 anti CD40 + DKW133 141 acute rejection I/CAN I 86 Hd Ri279 222 231 acute rejectionI/CAN I-II CsA 97064 >307 alive and well; no acute rejection/CAN IRi301 >307 alive and well; no acute rejection/CAN II-IIILd = low dose;Hd = high dose;PNF: primary non-function of graft;CAN: chronic allograft ephropathy;CMV: cytomegalovirus virus infection

In group 3, in which the animals received ATG in addition toanti-CD40+86, rejection started before the end of the Mab treatment inspite of the fact that from day 42 onwards steroids and CsA were given.The median time to rejection was 42 days and this was, again,significantly different from the time to rejection in group 2 (Cox'sproportional hazard analysis) (See FIG. 2.)

In contrast, as shown in FIG. 3, none of the animals in group 4, whichreceived anti-CD40+anti-CD86 followed by CsA showed signs of transplantrejection during the treatment period. Two animals rejected after CsAtreatment was stopped (one on day 141 and one on day 231), but twoanimals were still alive at the end of the observation period (>700 dayspost transplantation). Thus, the biopsies confirmed that the time torejection was significantly longer in all animals in group 4 than thetime to rejection in all other groups. Moreover, these results confirmedthat long-term survival and graft-tolerance has been achieved in someanimals even in the absence of continuous immunosuppressive drugtreatment.

Example 4 Host Immune Response to the Therapeutic Mabs

The production of host antibodies against ch5D12 and chFun-1 weredetermined in order to evaluate the host immune response to thesetherapeutic Mabs.

In animals treated with anti-CD40 alone (group 1) and withanti-CD40+antiCD-86 (group 2), three animals were killed before any RACAresponse could be detected. Two animals from group 1, with graftsurvival times of 42 and 217 days, had positive anti-ch5D12 IgM RACAstarting on days 13 and 11, respectively. These reactions persisted formore than a week. None of the animals from group 2 had a positiveanti-ch5D12 IgM response, but three animals had rather low, butpositive, anti-Fun-1 IgM indexes, all starting on day 11.

In both groups 1 and 2, a number of animals developed anti-ch5D12 IgGresponses. One animal from group 1 developed a relatively lowanti-ch5D12 IgG titer, more than 10 days after the last injection on day56, and had a graft survival of 91 days. The other two animals in group1 (graft survivals of 135 and 217 days) did not develop RACA. Animals ingroup 2 that rejected during treatment had high titers within 4 weeksafter the start of treatment. Because of this anti-ch5D12 RACAdevelopment, these two animals had rapidly declining levels of ch5D12(see below) and rejected early. Another two animals from group 2developed anti-ch5D12 IgG antibodies immediately after treatment wasstopped, and these animals rejected on days 71 (Grade I) and 78 (gradeI-II). One animal in this group developed a lower anti-ch5D12 IgG titerand rejected on day 116. Two animals did not develop a RACA responseagainst ch5D12, and these animals rejected on days 61 (fade II-IIIacute+0-−I chronic) and 75 (grade I). In general, it appears that forgroups 1 and 2, animals that did not develop any anti-ch5D12 IgG titergenerally survived longer than animals that did develop an anti-ch5D12IgG RACA response.

chFun-1 levels in animals of group 3 all showed a significant drop byday 20 to 30, which could be explained by the anti-chFun-1 responsepresent in all animals. ch5D12 levels in animals of group 3 also showeda significant drop. With the exception of Ri251, all animals werenegative for an anti-ch5D12 response. Although the absorbance wasincreased in some post-transplantation samples, this never rose abovepre-value+3×SD, with Ri251 as an exception.

Considerable anti-chFun-1 antibodies were also found in the animals ofgroup 4. The two animals with the highest titers of this Mab in thisgroup (97064 and Ri279) also had the lowest ch5D12 levels. This resultsshow that chFun-1 is more immunogenic than ch5D12 in rhesus monkeys andfurthermore results for group 4 show that in this group anti-chFun-1antibody response does not induce rejection as shown by the longsurvival time of all animals in this group.

Example 5 Correlation of Therapeutic Mab Levels and Graft Survival

The serum Mab levels of the therapeutic Mabs in each animal weredetermined to examine the correlation of Mab concentration and graftsurvival.

Circulating Mab levels in these the low dose animals for groups 1a and2a were found to be lower than 100 μg/ml serum after day 14. Therefore,the remaining animals in these groups were treated with a doubling ofthe dosing schedule to try and maintain circulating Mab levels above 100μg/ml throughout the treatment period. Animals in group 1b, and oneanimal in group 1b that developed a RACA response against ch5D12demonstrated ch5D12 levels of less than 100 μg/ml with rapidly declininglevels thereafter. These animals rejected early (days 8, 30, 42). Therest of the animals in group 1b and in group 2 that maintained highercirculating levels of ch5D12 had longer survival rates.

Some of the animals in group 3 were found to have low Mab levels at theday 0, and in all animals, levels were already below 100 μg/ml serum byday 40. This was the case for both ch5D12 and chFun-1Mabs. The twoanimals that lived the longest (Ri139 and Ri204), had at least a lowlevel of both Mabs in their blood around day 40, while the animals thatlived a shorter time had almost no Mabs in their blood by day 40.

In striking contrast, the Mab levels in animals in group 4 stayed highuntil the end of the treatment period. However, in this group, nocorrelation could be found between the height of the Mab levels and thesurvival time. Specifically, animal 97064 had the lowest levels of bothMabs, and this animal lived longer than at least two other monkeys (DKWand Ri279).

Example 6 Correlation of Donor Specific Antibody Levels and GraftRejection

Donor-specific antibodies were also measured as an indicator of graftrejection in all animals, except animal C146.

Donor-specific IgG antibodies developed in three animals of group 1(96087, Ri149 and DXW). In all these cases the antibodies only reachedsignificant levels at the day of rejection. Even then, the percentage ofdonor cells stained, was lower than 20%. Three animals of group 3(Ri251, Ri139 and Ri203) developed donor-specific IgG antibodies. InRi251 and Ri203 this was correlated with a short survival time.Generally, donor-specific antibodies are not known to have a detrimentaleffect on survival, but can be an indicator of the poorimmunosuppressive state that the animals are in.

None of the animals of groups 2 and 4 developed anti-donor IgGantibodies. The anti-donor IgM antibodies were difficult to interpretbecause of a high background that varied per test. However, some animals(e.g., Ri140, group 3) formed donor-specific IgM antibodies, while noIgG antibodies could be detected.

Example 7 Immunophenotyping of Peripheral Blood Lymphocytes

To investigate the systemic effects of the Mab treatments, lymphocytesubset FACS analyses were performed at regular time points using wholeEDTA blood.

As demonstrated in FIG. 5, cells from animals of group 2 and 3 could notbe stained using 5D12/FITC during treatment, but were detectable usinganother, non-competing anti-CD40 Mab. This indicates that CD40 wascompletely coated with ch5D12 in vivo, but that CD40 positive cells werenot removed from the circulation, although a small decrease in CD40positive cells can be seen from day 7 until day 28.

FIG. 6 shows the CD86 expression on the cells of the animals treatedwith the combination of ch5D12 and chFun-1. The anti-CD86 mAb stainedmore cells than Fun-1. As for ch5D12, no cells could be stained byFun-1/FITC and a decrease in CD86 positive cells was observed,indicating both a complete coating of CD86 and down-regulation of thenumber of CD86 positive cells. CD3⁺, CD4⁺, CD8⁺, and CD20⁺ cellpopulations did not change during the time of treatment.

The animals in group 3, treated with ATG, showed upon the return of thelymphocytes a preferential return of CD8+ cells. This could be anexplanation of the early rejection as CD8+ T cells are thought to beresponsible for cytolysis while regulatory T cells are of the CD4+phenotype (See FIG. 7).

These results clearly show full coating of both CD40- and CD86-bearingcells. Furthermore Mab treatment was without an effect on the number ofvarious immune cells in the circulation, showing that the Mabs did notcause cell depletion. ATG pre-treatment caused depletion of T cells,showing first CD8+ re-appearance in the absence of CD4+ regulatorycells.

Example 8 Latent TGF-β

In this example, development of TGF-β was studied as evidence oftolerance to transplant. Torrealba et al. (2004) have found that latentTGF-β in biopsies of stable kidney graft recipients correlates with theabsence of rejection and anti-donor responses in the trans-vivo DTHassay.

Kidney biopsies taken from monkeys in groups 1, 2, and 3 were analysedfor the presence of latent TGF-β. Biopsies were taken during treatment,as well as post-treatment. Kidney biopsies were stained for latent TGF-βand scored blindly for the number of TGF-β positive areas per tubule.FIG. 8 shows mean latent TGF-β staining/tubulus per group (+/−SEM).Latent TGF-beta is absent at the time of rejection, when euthanasia isindicated. Biopsies taken during costimulation blockade also have onlylow amounts of latent TGF-β present in the graft. A trend can be seenthat latent TGF-β expression is decreased in the group of animals thatreject after cessation of treatment (group 2), as compared to group 1,while no differences in Banff rejection score could be detected betweenboth groups. Animals of group 3 have lower amounts of latent TGF-β thananimals both in groups 1 and 2 in day 70 and day 112 biopsies. Thetreatment with CsA seems to cause these lower levels of TGF-β, but afterCsA is stopped, levels of TGF-β staining increase. The development ofTGF-β staining during the post transplant period was shown by biopsiesof two long-term surviving monkeys (>1130 and >1160 days). Earlybiopsies demonstrate a pattern of isolated areas of staining in theinterstitium, while later biopsies demonstrate more widely dispersedareas of interstitial staining. The presence of TGF-β indicates thatactive down-regulation of immune reactivity may be one of the mechanismsby which graft rejection is prevented.

Discussion

As presented above, six different treatment regimens were tested inrhesus monkeys that underwent kidney allograft transplantation: (1)anti-CD40 low dose (Group 1a); (2) anti-CD40 high dose (Group 1b);anti-CD86 low dose (Group 2a); (4) anti-CD86 high dose (Group 2b); (5)Pre-treatment with ATG, followed by anti-CD40 and anti-CD86 high dose,and then steroids and CsA (Group 3); and (6) anti-CD40 and anti-CD86high dose, followed by CsA (Group 4).

Treatment with anti-CD40 Mabs provided an immunosuppressive effect forkidney allografts in rhesus monkeys treated with high doses of the Mabs(group 1b). Although significant numbers of monocytes were seen in graftbiopsies on days 21 and 42, no impairment of graft function was found.However, after discontinuation of Mab treatment, rejection occurred in 3out of 4 animals (1 died of other causes), and two animals showed signsof chronic graft rejection. This is likely that indication that theblockade of CD40 has resulted in immune suppression mediated byregulatory T cells, which disappeared after discontinuation of Mabtreatment.

The combination of anti-CD40 plus anti-CD86 also prevented transplantrejection during Mab treatment, although in one case the rejectionprocess was already ongoing during the last weeks of Mab treatment.However, as with the anti-CD40 treatment, all animals rejected thekidney allografts in an acute fashion shortly after discontinuation ofthe treatment. Therefore, it seems that if anti-CD40 promoted theappearance of regulatory T cells, as evidenced by graft infiltratingcells, the anti-CD86 treatment may have counteracted this. Thistreatment is likely a more a general suppression of T-cell activationand once it is was stopped, the grafts are rejected.

The addition of ATG to the combination of anti-CD40 plus anti-CD86treatment resulted in an even earlier rejection than when anti-CD40 plusanti-CD86 was given alone. ATG results in a rigorous depletion of Tcells, both from the periphery as well as from central lymphoid tissue.This results in immunosuppression. T cells start to reappear 2 to 3weeks after the treatment, with CD8⁺ T cells reappearing earlier thatCD4⁺ T cells. This imbalance may be the cause of the earlier rejectionin this group. Rather than synergizing, ATG and the blockade ofco-stimulation thus appear to counteract one another, and should not becombined in protocols aiming at induction of graft prolongation wherethe formation of regulatory T cells is involved.

In contrast to the counteractive effects of calcineurin inhibitors onthe tolerizing potential of costimulation blockade described by others(see, e.g., Kirk et al., 1999), the data presented herein demonstratethat treatment with an anti-CD40 antagonist alone or in combination witha anti-CD86 antagonist, followed by CsA treatment, not only preventedgraft rejection during treatment, but achieved long-term survival andtransplant tolerance in some of the subjects. In addition, the datapresented herein demonstrate that by co-administering an anti-CD40antagonist alone or in combination with an anti-CD86 antagonist, thelevel of immunosuppressive drugs required for maintenance therapy waslower than that used in conventional immunosuppressant therapies. Forexample, while two of four animals subjected to the combined antagonistand immunosuppressive drug regiment rejected their transplant after CsAwas stopped, two animals have survived without a rise of serumcreatinine more than 100 weeks in the complete absence of any immunesuppressive maintenance therapy. Therefore, costimulation blockadefollowed by conventional immunosuppression significantly reduces theamount of immunosuppression needed to maintain graft survival.

REFERENCES

-   1. Bontrop R E, Otting N, Slierendregt B L, Lanchbury J S. Evolution    of major histocompatibility complex polymorphisms and T-cell    receptor diversity in primates. Immunol Rev 1995; 143: 33-62.-   2. Doxiadis G G, Otting N, de Groot N G, Noort R, Bontrop R E.    Unprecedented polymorphism of Mhc-DRB region configurations in    rhesus macaques. J Immunol 2000; 164 (6): 3193-9.-   3. Doxiadis G G, Otting N, Antunes S G, et al. Characterization of    the ABO blood group genes in macaques: evidence for convergent    evolution. Tissue Antigens 1998; 51 (4 Pt 1): 321-6.-   4. Fishman J A, Rubin R H. Infection in organ-transplant recipients.    N Engl J Med 1998; 338 (24): 1741-51.-   5. Haanstra K G, Ringers, J, Sick E A et al., Transplantation, 2003,    75 (5): 637-643.-   6. Hausen B, Klupp J, Christians U, et al. Coadministration of    either cyclosporine or steroids with humanized monoclonal antibodies    against CD80 and CD86 successfully prolong allograft survival after    life supporting renal transplantation in cynomolgus monkeys.    Transplantation 2001; 72 (6): 1128-37.-   7. Kenyon N S, Chatzipetrou M, Masetti M, et al. Long-term survival    and function of intrahepatic islet allografts in rhesus monkeys    treated with humanized anti-CD154. Proc Natl Acad Sci USA 1999; 96    (14): 8132-7.-   8. Kenyon N S, Fernandez L A, Lehmann R, et al. Long-term survival    and function of intrahepatic islet allografts in baboons treated    with humanized anti-CD154. Diabetes 1999; 48 (7): 1473-81.-   9. Kirk A D, Harlan D M, Armstrong N N, et al. CTLA4-Ig and    anti-CD40 ligand prevent renal allograft rejection in primates. Proc    Natl Acad Sci USA 1997; 94 (16): 8789-94.-   10. Kirk A D, Burkly L C, Batty D S, et al. Treatment with humanized    monoclonal antibody against CD154 prevents acute renal allograft    rejection in nonhuman primates. Nat Med 1999; 5 (6): 686-93.-   11. Kirk A D, Tadaki D K, Celniker A, et al. Induction therapy with    monoclonal antibodies specific for CD80 and CD86 delays the onset of    acute renal allograft rejection in non-human primates.    Transplantation 2001; 72 (3): 377-84.-   12. Knechtle S J, Harnawy M M, Hu H, Fechner Jr J H, Cho C S.    Tolerance and near-tolerance strategies in monkeys and their    application to human renal transplantation. Immunol Rev 2001; 183:    205-13.-   13. Montgomery S P, Hale D A, Hirshberg B, Harlan D M, Kirk A D.    Preclinical evaluation of tolerance induction protocols and islet    transplantation in non-human primates. Immunol Rev 2001; 183:    214-22.-   14. Neuhaus P, Neuhaus R, Wiersema H D, Borleffs J C, Balner H. The    technique of kidney transplantation in rhesus monkeys. J Med    Primatol 1982; 11 (3): 155-62.-   15. Ossevoort M A, Ringers J, Kuhn E M, et al. Prevention of renal    allograft rejection in primates by blocking the B7/CD28 pathway.    Transplantation 1999; 68 (7): 1010-8.-   16. Penn I. Post-transplant malignancy: the role of    immunosuppression. Drug Saf 2000; 23 (2): 101-13.-   17. Pierson R N, 3rd, Chang A C, Blum M G, et al. Prolongation of    primate cardiac allograft survival by treatment with ANTI-CD40    ligand (CD154) antibody. Transplantation 1999; 68 (11): 1800-5.-   18. Racusen L C, Solez K, Colvin R B, et al. The Banff 97 working    classification of renal allograft pathology. Kidney Int 1999; 55    (2): 713-23.-   19. Torrealba J R, Katayama M, Fechner J H, Jr., et al. Metastable    tolerance to rhesus monkey renal transplants is correlated with    allograft TGF-beta 1+CD4+T regulatory cell infiltrates. J Immunol    2004; 172 (9): 5753.

1-25. (canceled)
 26. A method of inducing tolerance to a transplant in asubject comprising: (a) administering multiple doses of atherapeutically effective amount of a CD40 antagonist, alone or incombination with a CD86 antagonist, wherein the first dose of antagonistis given before or at the time of transplantation; and (b) administeringmultiple doses of a therapeutically effective amount of animmunosuppressive drug, wherein the first dose of the immunosuppressivedrug is given at least about 2 days after transplantation.
 27. Themethod of claim 26, wherein the CD40 antagonist is administered for aperiod sufficient to tolerize T cells to the transplant.
 28. The methodof claim 26, wherein the CD40 antagonist is administered for a period ofat least about 6-12 weeks.
 29. The method of claim 26, wherein the CD40antagonist is administered in an amount sufficient to achieve a serumlevel of at least about 10-300 μg/ml.
 30. The method of claim 26,further comprising administering an antagonist ex vivo to the transplantprior to transplantation.
 31. The method of claim 26, wherein the firstdose of the immunosuppressive drug is administered at least about 5 daysafter transplantation.
 32. The method of claim 26, wherein the firstdose of the immunosuppressive drug is administered at least about 2weeks after transplantation.
 33. The method of claim 26, wherein thefirst dose of the immunosuppressive drug is administered followingcompletion of administration of the antagonist.
 34. The method of claim26, wherein the first dose of the immunosuppressive drug is administeredupon the onset of transplant rejection.
 35. The method of claim 26,wherein the immunosuppressive drug is administered for a period untiltolerance to the transplant is achieved in the absence of the antagonistor the immunosuppressive drug.
 36. The method of claim 26, wherein theimmunosuppressive drug is administered for a period of at least about4-12 weeks.
 37. The method of claim 26, wherein the antagonist and theimmunosuppressive drug are administered in tapering dosages.
 38. Themethod of claim 26, wherein administering antagonist in step (a)comprises administering a combination of the CD40 antagonist and theCD86 antagonist.
 39. The method of claim 26, wherein the antagonist isselected from the group consisting of an antibody, a bispecificantibody, a soluble receptor, a peptide and a small molecule.
 40. Themethod of claim 39, wherein the antibody is selected from the groupconsisting of a chimeric antibody, a humanized antibody and a humanantibody.
 41. The method of claim 26, wherein the CD40 antagonist isch5D12.
 42. The method of claim 38, wherein the CD40 antagonist isch5D12 and the CD86 antagonist is chFun-1.
 43. The method of claim 26,wherein the immunosuppressive drug is a signal 1 blocker.
 44. The methodof claim 26, wherein the immunosuppressive drug is selected from thegroup consisting of cyclosporine, tacrolimus, azathioprine, acorticosteroid, mycophenolate mofetil, rapamycin, OKT3 and an anti-CD25antibody.
 45. The method of claim 26, wherein the immunosuppressive drugis cyclosporine A.
 46. The method of claim 26, wherein the transplant isan allograft.
 47. The method of claim 26, wherein the transplant is anorgan.
 48. A method of inducing tolerance to a transplant in a subjectcomprising: (a) administering a therapeutically effective amount of aCD40 antagonist, alone or in combination with a CD86 antagonist, over aperiod of at least about 4-10 weeks, wherein the first dose ofantagonist occurs before or at the time of transplantation; and (b)administering a therapeutically effective amount of an immunosuppressivedrug over a period sufficient to achieve tolerance to the transplant inthe absence of antagonist or the immunosuppressive drug, wherein thefirst dose of the immunosuppressive drug occurs no sooner than about 4weeks after transplantation.
 49. A method of inducing tolerance to atransplant in a subject comprising: (a) administering multiple doses ofa therapeutically effective amount of a CD40 antagonist, alone or incombination with an antagonist of a second costimulatory molecule,wherein the first dose of antagonist is given before or at the time oftransplantation; and (b) administering multiple doses of atherapeutically effective amount of an immunosuppressive drug, whereinthe first dose of the immunosuppressive drug is given at least about 2days after transplantation.
 50. The method of claim 49, wherein theantagonist of the second costimulatory molecule is a CD86 antagonist.